Methods and materials for conferring resistance to pests and pathogens of plants

ABSTRACT

Methods and materials for conferring pest resistance to plants are provided. Plants are transformed with a silencing construct homologous to a gene of a plant pest that is essential for the survival, development, or pathogenicity of the pest. This results in the plant producing RNAi to the selected gene, which, when ingested by the pest results in silencing of the gene and a subsequent reduction of the pest&#39;s ability to harm the plant. In other embodiments, the pest&#39;s reduced ability to harm the plant is passed on to pest progeny. Methods and materials for depathogenesis of pests is also provided.

This Application claims priority to U.S. Provisional Application Ser.No. 60/621,542 filed 21 Oct. 2004 and U.S. Provisional Application Ser.No. 60/657,821 filed 1 Mar. 2005 both of which are hereby incorporatedby reference herein in their entirety.

FIELD OF THE INVENTION

Plant pests (e.g. fungal pathogens, bacteria, nematodes, insects,viruses, etc.) cause major losses of food and fiber throughout theworld, especially in developing countries. Losses include directproduction or pre-harvest losses, postharvest and storage losses, andthe diminution of food quality and safety (e.g. by production ofmycotoxins).

Other resultant losses from plant pests are observed in plants valuedfor aesthetic, olfactory, or ecologic properties.

Plant pests can sometimes be controlled by application of chemicals(e.g. fungicides, insecticides, nematicides, parasiticidals),manipulation or management of the microenvironment or by genes forresistance to the pathogen.

Discovery and introduction of a “new” gene for resistance frequentlycauses the development or selection of a new race of the pathogen ableto infect plants containing that “new” gene. This has best beendemonstrated by the rusts and smuts of cereal crops, but it also occurswith soil borne diseases such as black shank of tobacco and root andstem rot of soybean, caused by Phytophthora nicotianae and P. sojae,respectively. There are at least two races of P. nicotianae and morethan 70 races of P. sojae, all requiring different genes or combinationsof genes for disease resistance.

The fungal genus Phytophthora comprises many species of very destructivepathogens which cause serious diseases of plants. These include blights,damping-offs, cankers, fruit rots, root rots, wilts, and many othersymptoms that affect a wide variety of food, fiber and oil cropsincluding avocado, cacao, canola, citrus, pepper, potato, soybean,tobacco, tomato, pine, rubber, oak trees, etc.

In the past decade the phenomenon of gene silencing or RNA interference(RNAi) has been described and characterized in organisms as diverse asplants, fungi, nematodes, hydra and humans (Zamore and Haley, 2005). Itis considered to be an ancient defense mechanism wherein the hostorganism recognizes as foreign a double-stranded RNA molecule andhydrolyzes it. The resulting hydrolysis products are small RNA fragmentsof 21-30 nucleotides in length, called small interfering RNAs (siRNAs).The siRNAs then diffuse or are carried throughout the host, where theyhybridize to the mRNA for that specific gene and cause its hydrolysis toproduce more siRNAs. This process is repeated each time the siRNAhybridizes to its homologous mRNA, effectively preventing that mRNA frombeing translated, and thus “silencing” the expression of that specificgene.

Fire et al. (2003) describe a method of administering RNA to a hostwherein the RNA is composed of sense and antisense sequences homologousto a gene of that host cell to silence that host cell's gene. U.S. Pat.No. 6,506,559.

Results by van West et al. (1999) demonstrated internuclear genesilencing in Phytophthora infestans. They transformed P. infestans withthe infl elicitin gene in both the sense and antisense orientations.This resulted in silencing of both of the transgenes, as well as theendogenous gene. By somatic fusion of a silenced transgenic strain and awild-type strain, they demonstrated two essential points; first, thatthe presence of the transgene itself is not necessary to maintain thesilenced state; and second, the involvement of a trans-acting factorcapable of transferring the silencing signal between nuclei, later foundto be the siRNAs.

Waterhouse et al. (WO9953050A1) have described methods of conferringviral resistance to a plant by transforming the plant with a genesilencing construct directed towards a plant viral gene.

Wang et al. (US20030180945A1) describe a method of gene silencing in afungus by providing the fungus with a gene silencing construct directedtowards a fungal gene.

What is needed in the art is a plant resistance method that is readilyadaptable to changes in the biological environment such as a new andserious plant disease. For example, there were no occurrences of Asianrust in soybean in the USA until November 2004. Asian rust is predictedto cause losses estimated at 1 to 7 billion dollars (USDA) in 2005-2006.

The art would also be substantially advanced if there existed aversatile genetic method of conferring resistance to a plant against aspectrum of fungi, insects, nematodes, bacteria, etc.

SUMMARY OF THE INVENTION

There is now provided in the present invention a method and constructsfor conferring pest resistance to a plant comprising the step oftransforming a host plant cell with a heterologous polynucleotide, saidheterologous polynucleotide comprising:

(a) an antisense sequence having homology to a pest pathogenicity gene,and

(b) a sense sequence substantially complementary to said antisensesequence;

wherein a transcript of the heterologous polynucleotide is capable ofhybridizing to form a double-stranded region comprising nucleotidesencoded by the antisense sequence and the sense sequence;

wherein said sense and antisense sequences are each at least about 19nucleotides in length; and

wherein the antisense sequence is homologous to the pathogenicity geneof at least two pests from different species whereby the plant isresistant to said two pests as compared to substantially the same plantlacking the heterologous polynucleotide.

In another embodiment, the present invention provides a method andconstructs for conferring pest resistance to a plant comprising a stepof transforming a host plant cell with a heterologous polynucleotide,said heterologous polynucleotide comprising:

-   -   (a) a first antisense sequence having homology to a first pest        pathogenicity gene;    -   (b) a second antisense sequence having homology to a second pest        pathogenicity gene;    -   (c) a first sense sequence substantially complementary to said        first antisense sequence; and    -   (d) a second sense sequence substantially complementary to said        second antisense sequence,        wherein a transcript of the heterologous polynucleotide is        capable of hybridizing to form a double-stranded region        comprising nucleotides encoded by the first antisense sequence        and the first sense sequence,

wherein a transcript of the heterologous polynucleotide is capable ofhybridizing to form a double-stranded region comprising nucleotidesencoded by the second antisense sequence and the second sense sequence,and

wherein said first and second sense sequences and first and secondantisense sequences are each at least about 19 nucleotides in length.

Accordingly, a plant can now be made resistant to a plurality of pestsbelonging to the same or to different members selected from the groupconsisting of insects, bacteria, fungi, plants, and nematodes.

In another embodiment, the present invention provides a method andconstructs for conferring pest resistance to a plant comprising a stepof transforming a host plant cell with a heterologous polynucleotide,said heterologous polynucleotide comprising:

-   -   (a) a first promoter operably linked to an antisense sequence        wherein said antisense sequence is homologous to a pest        pathogenicity gene and operably linked to a terminator;    -   (b) a second promoter operably linked to a sense sequence        wherein said sense sequence is substantially complementary to        said antisense sequence and operably linked to a terminator;

wherein said second promoter is a strong promoter relative to the firstpromoter,

wherein a transcript of the heterologous polynucleotide is capable ofhybridizing to form a double-stranded region between sequences encodedby the antisense sequence and the sense sequence,

wherein said sense and antisense sequences are each at least about 19nucleotides in length.

In another embodiment, the present invention provides a method andconstructs for conferring pest resistance to a plant comprising the stepof transforming a host plant cell with a heterologous polynucleotide,said heterologous polynucleotide comprising:

(a) an antisense sequence having homology to a pest pathogenicity gene,and

(b) a sense sequence substantially complementary to said antisensesequence,

wherein a transcript of the heterologous polynucleotide is capable ofhybridizing to form a double-stranded region comprising nucleotidesencoded by the antisense sequence and the sense sequence,

wherein said sense and antisense sequences are each at least about 19nucleotides in length,

wherein the pest is selected from the group consisting of insects,bacteria, fungi, and nematodes, and

wherein the pest pathogenicity gene and the pest are selected from thosetaught herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the genetic map of the pCAMBIA1201 plant transformationplasmid used as an example throughout this teaching and which is thebackbone of pVZA100, pVZA200, pVZA300 and pVZA400.

FIG. 2 is a drawing of the insert in the intermediary plasmid pVZA1.

FIG. 3 is the pVZA100 silencing cassette cloned in plasmid pVZA3 andready for transfer to pCAMBIA1201.

FIG. 4 is autoradiograms showing the siRNAs isolated from transgenictobacco and from transgenic Phytophthora nicotianae. FIGS. 4A and 4Bshow the results of hybridization of the cutinase gene probe to siRNAsfrom wild type and transgenic tobacco plants and P. nicotianae,respectively.

FIG. 5 shows plant reactions to a natural epidemic of the blue molddisease of tobacco caused by Peronospora tabacina in the greenhouse.

FIG. 6 shows shoots emerging from transgenic soybean embryos.

FIG. 7 shows the results of soybean transformed with pVZA100. FIG. 7Ashows transformed soybean calli. FIG. 7B show GUS and hygromycinphosphotransferase genes detected in the transformed soybean callus.

FIG. 8 shows roots and minitubers produced in the culture plates ofpotato cells transformed with pVZA100.

FIG. 9 shows a polycistronic gene silencing construct capable ofsilencing essential genes in three fungal species causing seriousdiseases of soybeans.

FIG. 10 shows gene silencing constructs based on rDNA and capable ofsilencing these essential genes in Phytophthora, in Phakopsora and inboth species.

FIG. 11 shows a wild type tobacco plant (A) and a pVZA100 transgenictobacco plant (B), susceptible and resistant to Phytophthora nicotianae,respectively.

FIG. 12 is an autoradiogram showing the siRNAs isolated from atransgenic tobacco plant (3) and from Phytophthora nicotianae growing ona silenced transgenic tobacco plant (4).

FIG. 13 shows transgenic soybean plants resistant to Phytophthora sojae.

FIG. 14 shows the effects of pVZA300 and pVZA400 on the growth anddevelopment of transgenic Phytophthora nicotianae and Phytophthorasojae.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the PCR primer VZA 1F as used according to the subjectinvention.

SEQ ID NO: 2 is the PCR primer VZA 2R as used according to the subjectinvention.

SEQ ID NO: 3 is the PCR primer VZA 3F as used according to the subjectinvention.

SEQ ID NO: 4 is the PCR primer VZA 4R as used according to the subjectinvention.

SEQ ID NO: 5 is the silencing construct pVZA100 used according to thesubject invention.

SEQ ID NO: 6 is the silencing construct pVZA200 used according to thesubject invention.

SEQ ID NO: 7 is the silencing construct pVZA300 used according to thesubject invention.

SEQ ID NO: 8 is the silencing construct pVZA400 used according to thesubject invention.

SEQ ID NO: 9 is the forward PCR primer GUS as used according to thesubject invention.

SEQ ID NO: 10 is the reverse PCR primer GUS as used according to thesubject invention.

SEQ ID NO: 11 is the PCR primer pVZA100 and hygromycin phosphotrasferaseas used according to the subject invention.

SEQ ID NO: 12 is the PCR primer pVZA100 and hygromycin phosphotrasferaseas used according to the subject invention.

SEQ ID NO. 13 is the primer VZA 1 65R as used according to the subjectinvention.

SEQ ID NO. 14 is the primer VZA 2 69F as used according to the subjectinvention.

SEQ ID NO. 15 is the primer VZA 3 73F as used according to the subjectinvention.

SEQ ID NO. 16 is the primer VZA 3 73F as used according to the subjectinvention.

SEQ ID NO. 17 is the primer VZA 1 65 as used according to the subjectinvention.

SEQ ID NO. 18 is the primer VZA 6 70F as used according to the subjectinvention.

SEQ ID NO. 19 is the PCR primer VZA 7 72F as used according to thesubject invention.

SEQ ID NO. 20 is the PCR primer VZA 8 79F as used according to thesubject invention.

SEQ ID NO. 21 is the PCR VZA 9 84F primer as used according to thesubject invention.

SEQ ID NO. 22 is the PCR VZA 10 87F primer as used according to thesubject invention.

SEQ ID NO. 23 is the PCR VZA 11 89F primer as used according to thesubject invention.

SEQ ID NO. 24 is the PCR VZA 12 90F primer as used according to thesubject invention.

SEQ ID NO. 25 is the PCR VZA 13 91F primer as used according to thesubject invention.

SEQ ID NO. 26 is the PCR VZA 14 81F primer as used according to thesubject invention.

SEQ ID NO. 27 is the PCR VZA 15 82F primer as used according to thesubject invention.

SEQ ID NO. 28 is the PCR primer VZA 6 83F as used according to thesubject invention.

SEQ ID NO. 29 is the PCR primer VZA 17 85F as used according to thesubject invention.

SEQ ID NO. 30 is the PCR VZA 0.18 86F primer as used according to thesubject invention.

SEQ ID NO. 31 is the PCR VZA 19 64R primer as used according to thesubject invention.

SEQ ID NO. 32 is the PCR VZA 20 77F primer as used according to thesubject invention.

SEQ ID NO. 33 is the PCR VZA 21 63F primer as used according to thesubject invention.

SEQ ID NO. 34 is the PCR VZA 22 66R primer as used according to thesubject invention.

SEQ ID NO. 35 is the PCR VZA 23 80F primer as used according to thesubject invention.

SEQ ID NO. 36 is the PCR VZA 24 92F primer as used according to thesubject invention.

SEQ ID NO. 37 is the PCR primer VZA 25 93F as used according to thesubject invention.

SEQ ID NO. 38 is the PCR primer VZA 2R as used according to the subjectinvention.

SEQ ID NO. 39 is the PCR primer cathepsin as used according to thesubject invention.

SEQ ID NO. 40 is the PCR primer elicitin as used according to thesubject invention.

SEQ ID NO. 41 is the PCR primer rDNA FP as used according to the subjectinvention.

SEQ ID NO. 42 is the PCR primer rDNA RP as used according to the subjectinvention.

SEQ ID NO. 43 is the marker Phialophora gregata ribosomal RNA gene(rDNA) as used according to the subject invention.

SEQ ID NO. 44 is the genotype B DNA marker Phialophora gregata ribosomalRNA gene (rDNA) as used according to the subject invention.

SEQ ID NO. 45 is the polymerase II subunit Sclerotinia sclerotiorumpartial rpb2 gene for RNA as used according to the subject invention.

SEQ ID NO. 46 is the ribosomal RNA gene Puccinia hordei 18S as usedaccording to the subject invention.

SEQ ID NO. 47 is the hexose transporter (hxt2) gene Sclerotiniasclerotiorum as used according to the subject invention.

SEQ ID NO. 48 is Fusarium solani pisi cutinase mRNA as used according tothe subject invention.

SEQ ID NO. 49 is the genome or conserved protein motif of Phytophthoranicotianae as used according to the subject invention.

SEQ ID NO. 50 is a ribosomal DNA sequence found in the Phakopsorapachyrhizi genome as used according to the subject invention.

SEQ ID NO. 51 is a Fusarium sambucinum translation elongation factor 1alpha as used according to the subject invention.

SEQ ID NO. 52 is an elicitin gene sequence found in the Phytophthorainfestans genome as used according to the subject invention.

SEQ ID NO. 53 is a partial rpb2 gene for RNA polymerase II subunitSclerotinia sclerotiorum as used according to the subject invention.

SEQ ID NO. 54 is rRNA Stenocarpella maydis as used according to thesubject invention.

SEQ ID NO. 55 is the 18S ribosomal RNA from a Cercospora zeae-maydisGroup II as used according to the subject invention.

SEQ ID NO. 56 is Cathepsin B protease from Myzus persicae as usedaccording to the subject invention.

SEQ ID NO. 57 is cysteine protease from Leptinotarsa decemlineata asused according to the subject invention.

SEQ ID NO. 58 is Verticillium dahliae rDNA as used according to thesubject invention.

SEQ ID NO. 59 is partial sequence of 5.8S ribosomal RNA found in thePeronospora berteroae genome as used according to the subject invention.

SEQ ID NO. 60 is a genome subunit of cytochrome oxidase Meloidogynechitwoodi as used according to the subject invention.

SEQ ID NO. 61 is ribosomal RNA P. scribneri as used according to thesubject invention.

SEQ ID NO. 62 is ribosomal RNA Heterodera glycines as used according tothe subject invention.

SEQ ID NO. 63 is RNA polymerase II sequence Magnaporthe grisea as usedaccording to the subject invention.

SEQ ID NO. 64 is rRNA Puccinia hordei 18S as used according to thesubject invention.

SEQ ID NO. 65 is the partial sequence 5.8S ribosomal RNAPseudoperonospora humuli as used according to the subject invention.

SEQ ID NO. 66 is Botrytis cinerea cytochrome P450 monoxygenase as usedaccording to the subject invention.

SEQ ID NO. 67 is rRNA Pseudomonas syingae as used according to thesubject invention.

SEQ ID NO. 68 is Clavibacter michiganense michiganense Cel A gene asused according to the subject invention.

SEQ ID NO. 69 is Clavibacter michiganense endo β-glucosidase gene asused according to the subject invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following definitions apply:

Cell (or host plant cell) means a cell or protoplast of a plant cell andincludes isolated cells and cells in a whole plant, plant organ, orfragment of a plant. It also includes non-isolated cells.

Double stranded region means a region of a polynucleotide wherein thenucleotides are capable of hydrogen bonding to each other. Such hydrogenbonding can be intramolecular or intermolecular (e.g. singletranscription unit forming a double stranded region with the so-calledhairpin or two transcription units that align appropriately forcomplementary sequences to hydrogen bond). To be a double strandedregion, according to the present invention, it is not necessary for 100%of the nucleotides to be complementary and hydrogen bonded within aregion. It is merely necessary for sufficient base pairing to occur togive the RNA a substantial double stranded character (e.g. an indicativemelting point).

Exogenous gene means a gene that is not normally present in a given hostgenome in the present form. In this respect, the gene itself may benative to the host genome, however the exogenous gene will comprise thenative gene altered by the addition or deletion of one or more differentregulatory elements or additional genes.

Gene or genes means nucleic acid sequences (including both RNA or DNA)that encode genetic information for the synthesis of a whole RNA, awhole protein, or any functional portion of such whole RNA or wholeprotein sufficient to possess a desired characteristic.

Heterologous polynucleotide means any polynucleotide that is not foundin a non-transformed host plant. A polynucleotide is not excluded frombeing a heterologous polynucleotide by the presence of endogenouspolynucleotide sequences.

Homologous, in the context of the relationship between the antisensesequence and a pest pathogenicity gene (i.e. the DNA strand that bindsRNA polymerase and directs transcription of the pest pathogenicity genemRNA which is the antisense strand), means having sequence similaritysufficient to allow hybridization in vivo, in vitro, and/or ex vivounder low stringency conditions between the antisense sequence and thepest pathogenicity gene mRNA.

Host plant cell resistance means that a plant is less susceptible to thedeleterious effects of a plant pest when compared to a host plant cellnot transformed with said construct.

Inhibition of gene expression means a decrease in the level of proteinand/or mRNA product from a target gene. The consequences of inhibitioncan be confirmed by examination of the outward properties of the cell ororganism (as presented below in the examples) or by biochemicaltechniques such as RNA solution hybridization, nuclease protection,Northern hybridization, polymerase chain reaction (PCR), reversetranscription (RT) reverse transcription PCR(RT/PCR), gene expressionmonitoring with a microarray, antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA),other immunoassays, and fluorescence activated cell analysis (FACS).

Marker gene means a gene that imparts a distinct phenotype to a cellexpressing the marker gene and thus allows such transformed cells to bedistinguished from cells that do not contain the gene.

Operably linked means joined as part of the same nucleic acid molecule,suitably positioned and oriented for transcription to be initiated fromthe promoter.

Pest pathogenicity gene means any pest gene that serves a role in such apest's deleterious effects on a host plant. Deleterious effects means,by way of example, a negative effect on plant growth (numbers and/orsize of the plant), development, quality, and/or reproduction. By way ofexample only, a pest pathogenicity gene may be one that serves a role inany of pest growth, development, replication and/or reproduction, orpest invasion or infection of the host plant.

Pathogenic, in reference to a pest, means a pest's ability to causedeleterious effects on a plant. Pathogenicity is augmented by any pestpathogenicity genes.

Plant pest means any living organism that is deleterious to plant growth(numbers and/or size of the plant), development, quality, yield, and/orreproduction. Non-limiting examples of such pests are insects, fungi,nematodes, bacteria, and viruses.

Quality means any aspect of a plant that can be considered desirable. Byway of non-limiting example, quality can refer to a plant's appearance,yield, flavor, nutritional value, aroma, or content.

Substantially complementary, with respect to the sense and antisensesequences means sufficiently complementary to allow for formation of adouble stranded molecule.

Transformation means a process of introducing an exogenous DNA sequence(e.g., a vector, a recombinant DNA molecule) into a cell or protoplastin which that exogenous DNA is incorporated into a chromosome or iscapable of autonomous replication in a manner such that a heterologousRNA is transcribed.

Transcript means RNA encoded by DNA. In context of sense and antisensetranscripts of the present invention, such sense and antisensetranscripts can be part of the same polynucleotide or they can be 2separate polynucleotides (i.e., each having its own 5′ and 3′ end).

Treating a plant pest means a method to reduce, eliminate, reverse, orprevent the deleterious effects of a plant pest on the plant.

Vector means a DNA molecule capable of replication in a host cell and/orto which another DNA segment can be operatively linked so as to bringabout replication of the attached segment. A plasmid is an exemplaryvector.

Underlying the various embodiments of the present invention is treatinga plant to confer pest resistance by transforming a host plant cell witha heterologous polynucleotide comprising:

(a) an antisense sequence having homology to a pest pathogenicity gene,and

(b) a sense sequence substantially complementary to said antisensesequence;

wherein said sense and antisense sequences are capable of hybridizing toeach other to form a double-stranded region and wherein said sense andantisense sequences are each at least about 19 nucleotides in length.

Without being bound by theory, it is believed that plants transformedaccording to the present invention transcribe an RNA molecule(s) with aregion homologous to and a region complementary to the pestpathogenicity gene, and wherein the transcript(s) form a double strandedRNA (dsRNA) molecule. The plant recognizes the dsRNA as a potentialforeign substance (e.g. a substance of viral origin). The dicer enzymeof the plant cuts the double stranded RNA into pieces of single-strandedRNA of about 23 nucleotides in length, called small interfering RNAs(siRNAs). These siRNAs are consumed by invading pests that have enteredthe plant via the digestion of plant cells (e.g. cutin). Once absorbed,the siRNAs can be incorporated into the pest's RNA-induced silencingcomplexes (RISC). The RISC complex can then digest the mRNA of thepest's homologous gene limiting the pest's ability to harm the plant.

In one embodiment, pest resistance is conferred to a plant comprisingthe step of transforming a host plant cell with a heterologouspolynucleotide, said heterologous polynucleotide comprising:

(a) an antisense sequence having homology to a pest pathogenicity gene,and

(b) a sense sequence substantially complementary to said antisensesequence,

wherein a transcript of the heterologous polynucleotide is capable ofhybridizing to form a double-stranded region comprising nucleotidesencoded by the antisense sequence and the sense sequence,

wherein said sense and antisense sequences are each at least about 19nucleotides in length,

wherein said pest pathogenicity gene is selected from the groupconsisting of cutinases, kinases, ribosomal RNAs, adhesins, elicitins,and G-proteins; and

wherein said pest is a fungus.

In one embodiment, pest resistance is conferred to a plant comprisingthe step of transforming a host plant cell with a heterologouspolynucleotide, said heterologous polynucleotide comprising:

(a) an antisense sequence having homology to a pest pathogenicity gene;and

(b) a sense sequence substantially complementary to said antisensesequence,

wherein a transcript of the heterologous polynucleotide is capable ofhybridizing to form a double-stranded region comprising nucleotidesencoded by the antisense sequence and the sense sequence,

wherein said sense and antisense sequences are each at least about 19nucleotides in length,

wherein said pest pathogenicity gene is selected from the groupconsisting of kinases, ribosomal RNAs, G-proteins, moulting factors,serine proteases, cysteine proteases, and juvenile hormone esterases;and

wherein said pest is an insect.

In one embodiment, pest resistance is conferred to a plant comprisingthe step of transforming a host plant cell with a heterologouspolynucleotide, said heterologous polynucleotide comprising:

(a) an antisense sequence having homology to a pest pathogenicity gene;and

(b) a sense sequence substantially complementary to said antisensesequence,

wherein a transcript of the heterologous polynucleotide is capable ofhybridizing to form a double-stranded region comprising nucleotidesencoded by the antisense sequence and the sense sequence,

wherein said sense and antisense sequences are each at least about 19nucleotides in length;

wherein said pest pathogenicity gene is selected from the groupconsisting of kinases, ribosomal RNAs, G-proteins, cuticle collagenproteins, and cathepsin proteases, and

wherein said pest is a nematode.

In one embodiment, pest resistance is conferred to a plant comprisingthe step of transforming a host plant cell with a heterologouspolynucleotide, said heterologous polynucleotide comprising:

(a) an antisense sequence having homology to a pest pathogenicity gene;and

(b) a sense sequence substantially complementary to said antisensesequence,

wherein a transcript of the heterologous polynucleotide is capable ofhybridizing to form a double-stranded region comprising nucleotidesencoded by the antisense sequence and the sense sequence,

wherein said sense and antisense sequences are each at least about 19nucleotides in length,

wherein said pest pathogenicity gene is selected from the groupconsisting of cutinases, mascerating enzymes, kinases, ribosomal RNAs,adhesins, and G-proteins; and

wherein said pest is a bacterium.

In one embodiment, the host plant is a soybean and the pest is onemember selected from the group consisting of Fusarium solani (e.g.,sudden death), Sclerotinia sclerotiorum (e.g., white mold), Phialophoragregata (e.g., brown stem rot), Phakospora pachyrhizi (e.g., Asian rust)and Phytophthora sojae (e.g., root and stem rot).

In one embodiment, the host plant is a cruciferous plant and the pest isAlubgo (e.g., white rust).

In one embodiment, the host plant is tobacco and the pest isPhytophthora (e.g., stem rot, root rot).

In one embodiment, the host plant is potato and the pest is Phytophthora(e.g., late blight).

In one embodiment, the host plant is broccoli and the is Pythium (e.g.,damping-off).

In one embodiment, the host plant is pea and/or sugar beet and the pestis Aphanomyces (e.g., root rot).

In one embodiment, the host plant is tobacco and the pest is Peronospora(e.g., blue mold).

It has further been discovered that gene silencing in the pest accordingto the present invention, can be conferred upon the progeny of thepest—progeny that never had direct contact with the transformed hostplant.

In one embodiment, the invention further comprises the step ofcultivating a pest with the transformed host plant cell.

In one embodiment, a transformed host plant cell of the presentinvention is co-cultivated with a pest.

Optionally, progeny of the pest are less pathogenic to a plant cellother than the transformed host plant cell.

In one embodiment, a plant regenerated from a host plant celltransformed according to the present invention, is cultivated in soilcontaminated with a pest.

In another embodiment, a plant regenerated from a host plant celltransformed according to the present invention, is cultivated in soil atrisk of being contaminated with a pest.

In another embodiment, a pest is co-cultivated with a plant regeneratedfrom a host plant cell transformed according to the present inventionand then cultivated in soil contaminated with a pest.

In another embodiment, a pest is co-cultivated with a plant regeneratedfrom a host plant cell transformed according to the present inventionand then cultivated in soil at risk of being contaminated with a pest.

In one embodiment, a pest is co-cultivated with the transformed plantand then cultivated in soil at risk of being contaminated with a pest.

Pest Pathogenicity Gene Homology

It has been further discovered that gene silencing according to thepresent invention can confer resistance to a surprisingly broad range ofpests and that exact identity between a region of the sense sequence andthe pest infectious gene is not required for the present invention to beeffective.

In one embodiment of the present invention, the pest pathogenicity geneis a gene that provides for cross-resistance. Such a pest gene isconserved within the pest phylum such that when used with the presentinvention, a plant is made resistant to a plurality of members of thepest phylum (i.e. cross-resistance). Moreover, pest pathogenicity genescan be chosen according to the present invention by bioinformatichomology analysis as known to those skilled in the art to providecross-resistance that is cross-species, cross-genus, cross-family, orcross-order resistance. For example, as will become clear from theexamples herein, a cutinase gene is useful in plants to conferresistance to a plurality of fungi species. The gene for rDNA is usefulin the pests of the present invention (i.e. insects, bacteria, fungi,and nematodes). Other non-limiting examples of such conserved genes arekinases, adhesions, G-proteins, elicitins, macerating enzymes, DNA andRNA polymerases, elongation factors, moulting factors, serine proteases,cysteine proteases, juvenile hormone esterase, cuticle collagenproteins, and cathepsin proteases and others set forth below. In anotherembodiment, a pest pathogenicity gene is selected to lack sufficienthomology with a plant gene to cause harmful effects of the heterologouspolynucleotide on the host plant (e.g. prevent silencing of a plantgene). For example, in one embodiment, the homology between theantisense sequence and the pest pathogenicity gene is less than about70%.

In one embodiment, the antisense sequence is homologous to the pestpathogenicity gene by at least about 70%.

Optionally, the homology is at least about 75%.

Optionally, the homology is at least about 80%.

Optionally, the homology is at least about 85%.

Optionally, the homology is at least about 90%.

Optionally, the homology is at least about 95%.

In one embodiment, a transcript of the sense sequence is homologous to atranscript of the pest pathogenicity gene (e.g. pest pathogenicity genemRNA) by at least about 70%.

Optionally, the homology is at least about 75%.

Optionally, the homology is at least about 80%.

Optionally, the homology is at least about 85%.

Optionally, the homology is at least about 90%.

Optionally, the homology is at least about 95%.

The above-mentioned homology can optionally extend over a stretch of atleast about 20 nucleotides, or at least about 25 nucleotides, or atleast about 50 nucleotides, or at least about 100 nucleotides.

Optionally, homology can be demonstrated between the antisense sequenceand the pest pathogenicity gene (i.e. the DNA strand that binds RNApolymerase and directs transcription of the pest pathogenicity genemRNA) in any of at least three ways.

(1) hybridization between the antisense sequence and correspondingregion of the pest gene sense strand.

(2) hybridization between the antisense transcript and the correspondingregion of the pest pathogenicity gene.

(3) hybridization between a transcript of a DNA complentary to theantisense sequence (i.e. antisense cDNA) and a corresponding region ofthe pest pathogenicity gene mRNA.

Hybridization, as set forth above, can be demonstrated under conditionsof low stringency.

Optionally, the conditions are ones of moderate to high stringency bytechniques well known in the art, as described, for example, in Keller,G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y.,pp. 169-170.

Examples of moderate to high stringency conditions are provided herein.Specifically, hybridization of immobilized DNA on Northern blots with32P-labeled gene-specific probes is performed using standard methods(Maniatis et al.). In general, hybridization and subsequent washes arecarried out under moderate to high stringency conditions that allowedfor detection of target sequences with homology to sequences exemplifiedherein. For double-stranded DNA gene probes, hybridization was carriedout overnight at 20-25° C. below the melting temperature (Tm) of the DNAhybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denaturedDNA. The melting temperature is described by the following formula fromBeltz et al. (1983): Tm=81.5° C.+16.6 Log [Na+]+0.41 (% G+C)-0.61(%formamide)-600/length of duplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

For oligonucleotide probes, hybridization was carried out overnight at10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes was determined by the following formula fromSuggs et al. (1981): Tm (° C.)=2(number T/A base pairs)+4(number G/Cbase pairs).

Washes were typically carried out as follows:

(1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1%SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment of greater than about 70 or so bases inlength, the following conditions can be used:

-   -   Low: 1 or 2×SSPE, room temperature    -   Low: 1 or 2×SSPE, 42° C.    -   Moderate: 0.2× or 1×SSPE, 65° C.    -   High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, polynucleotide sequencesuseful in the subject invention include mutations (both single andmultiple), deletions, and insertions in the described sequences, andcombinations thereof, wherein said mutations, insertions, and deletionspermit formation of stable hybrids with a target polynucleotide ofinterest. Mutations, insertions, and deletions can be produced in agiven polynucleotide sequence using standard methods known in the art.Other methods may become known in the future.

The mutational, insertional, and deletional variants of thepolynucleotide sequences of the invention can be used in the same manneras the exemplified polynucleotide sequences so long as the variants havesubstantial sequence similarity with the original sequence. As usedherein, substantial sequence similarity refers to the extent ofnucleotide similarity that is sufficient to enable the variantpolynucleotide to function in the same capacity as the originalsequence. Preferably, this similarity is greater than 75%; morepreferably, this similarity is greater than 90%; and most preferably,this similarity is greater than 95%. The degree of similarity needed forthe variant to function in its intended capacity will depend upon theintended use of the sequence. It is well within the skill of a persontrained in this art to make mutational, insertional, and deletionalmutations that are designed to improve the function of the sequence orotherwise provide a methodological advantage.

Vectors

Those skilled in the art are well able to construct vectors of thepresent invention (including those based on naked DNA) and designprotocols for recombinant gene expression. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al, 1989, Cold Spring Harbor Laboratory Press. Such applicabletechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Protocols in Molecular Biology,Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

Specific procedures and vectors previously used with wide success uponplants are described by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721),and Guerineau and Mullineaux, (1993). Plant transformation andexpression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed).Oxford, BIOS Scientific Publishers, pp 121-148.

Vectors according to the present invention may be provided isolatedand/or purified (i.e. from their natural environment), in substantiallypure or homogeneous form, or free or substantially free of other nucleicacid. Nucleic acid according to the present invention may be wholly orpartially synthetic.

Host Plants

The present invention may be used for transformation of any plant,including, but not limited to, corn (Zea mays), canola (Brassica napus,Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Cofea ssp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidental), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),oats, barley, vegetables, ornamentals, and conifers.

Optionally, plants of the present invention are crop plants (forexample, cereals and pulses, maize, wheat, potatoes, tapioca, rice,sorghum, millet, cassava, barley, pea, and other root, tuber, or seedcrops. Important seed crops for the present invention are oil-seed rape,sugar beet, maize, sunflower, soybean, and sorghum. Horticultural plantsto which the present invention may be applied may include lettuce,endive, and vegetable brassicas including cabbage, broccoli, andcauliflower, and carnations, geraniums, petunias, and begonias. Thepresent invention may be applied to tobacco, cucurbits, carrot,strawberry, sunflower, tomato, pepper, chrysanthemum, poplar,eucalyptus, and pine.

Optionally, plants of the present invention include grain seeds, such ascorn, wheat, barley, rice, sorghum, rye, etc.

Optionally, plants of the present invention include oil-seed plants. Oilseed plants include canola, cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc.

Optionally, plants of the present invention include leguminous plants.Leguminous plants include beans and peas. Beans include guar, locustbean, fenugreek, soybean, garden beans, cowpea, mung bean, lima bean,fava bean, lentils, chickpea, etc.

Host plants useful in the present invention are row crops and broadcastcrops. Non limiting examples of useful row crops are corn, soybeans,cotton, amaranth, vegetables, rice, sorghum, wheat, milo, barley,sunflower, durum, and oats.

Non-limiting examples of useful broadcast crops are sunflower, millet,rice, sorghum, wheat, milo, barley, durum, and oats.

Host plants useful in the present invention are monocots and dicots.

Non-limiting examples of useful monocots are rice, corn, wheat, palmtrees, turf grasses, barley, and oats.

Non-limiting examples of useful dicots are soybean, cotton, alfalfa,canola, flax, tomato, sugar beet, sunflower, potato, tobacco, corn,wheat, rice, lettuce, celery, cucumber, carrot, cauliflower, grape, andturf grasses.

Host plants useful in the present invention include plants cultivatedfor aesthetic or olfactory benefits. Non limiting examples includeflowering plants, trees, grasses, shade plants, and flowering andnon-flowering ornamental plants.

Host plants useful in the present invention include plants cultivatedfor nutritional value.

Host Cell Types

One skilled in the art will recognize the wide variety of host cellsthat can be contacted with the heterologous polynucleotides according tothe present invention. Non-limiting examples of such cells are those inembryogenic tissue, callus tissue types I, II, and III, hypocotyl,meristem, root tissue, tissues for expression in phloem, and the like.

Host plant cells can optionally be enriched for cells with a higherpotential to be transformed and/or a higher potential to regeneratemature plants. Manual selection of host plant cells, e.g., by selectingembryogenic cells from the surface of a Type II callus, is one meansthat may be used to enrich for host plant cells prior to culturing(whether cultured on solid media or in suspension). Optionally, cellscan be selected from those located at the surface of a cell cluster, andmay further be identifiable by their lack of differentiation, their sizeand dense cytoplasm. In one embodiment, host plant cells are lessdifferentiated, or not yet committed to differentiation. Thus, in oneembodiment, cells are identified and selected from those cells which arecytoplasmically dense, relatively unvacuolated with a high nucleus tocytoplasm ratio (e.g., determined by cytological observations), small insize (e.g., 10-20 μm), and capable of sustained divisions and somaticproembryo formation.

Optionally, host plant cells are identified through the use of dyes,such as Evan's blue, which are excluded by cells with relativelynon-permeable membranes, such as embryogenic cells, and taken up byrelatively differentiated cells such as root-like cells and snake cells(so-called due to their snake-like appearance).

Optionally, host plant cells are identified through the use of isozymemarkers of embryogenic cells, such as glutamate dehydrogenase, which canbe detected by cytochemical stains (Fransz et al., 1989). One skilled inthe art will recognize that isozyme markers such as glutamatedehydrogenase may lead to some degree of false positives fromnon-embryogenic cells such as rooty cells which nonetheless have arelatively high metabolic activity.

Almost all plant tissues may be transformed during dedifferentiationusing appropriate techniques described herein. Recipient cell targetsinclude, but are not limited to, meristem cells, Type I, Type II, andType III callus, immature embryos and gametic cells such as microspores,pollen, sperm, and egg cells. It is contemplated that any cell fromwhich a fertile plant may be regenerated is useful as a recipient cell.Type I, Type II, and Type III callus may be initiated from tissuesources including, but not limited to, immature embryos, immatureinflorescenses, seedling apical meristems, microspores, and the like.

Those cells that are capable of proliferating as callus also arerecipient cells for genetic transformation. The present inventionprovides techniques for transforming immature embryos and subsequentregeneration of fertile transgenic plants. Direct transformation ofimmature embryos obviates the need for long term development ofrecipient cell cultures. Pollen, as well as its precursor cells,microspores, may be capable of functioning as recipient cells forgenetic transformation, or as vectors to carry foreign DNA forincorporation during fertilization. Direct pollen transformation wouldobviate the need for cell culture.

Meristematic cells (i.e., plant cells capable of continual cell divisionand characterized by an undifferentiated cytological appearance,normally found at growing points or tissues in plants such as root tips,stem apices, lateral buds, etc.) may represent another type of recipientplant cell. Because of their undifferentiated growth and capacity fororgan differentiation and totipotency, a single transformed meristematiccell could be recovered as a whole transformed plant. In fact, it isproposed that embryogenic suspension cultures may be an in vitromeristematic cell system, retaining an ability for continued celldivision in an undifferentiated state, controlled by the mediaenvironment.

Pests

Plant pests useful in the present invention (i.e., can be renderednon-pathogenic according to the present invention), include fungi,nematodes, insects, bacteria, and parasitic plants such as striga,dodder and mistletoe.

Non-limiting examples of useful fungi include those set forth in Table4.

Non-limiting examples of such useful nematodes include those set forthin Table 3.

Non-limiting examples of such useful insects include aphids,leafhoppers, planthoppers, mealy bugs, and Lepidoptera larvae.

Plant pests usefully treated by the present invention include bacteria.Non-limiting examples of such bacteria are shown in Table 1.

Plant pests usefully treated by the present invention includes rusts.Non-limiting examples of such rust are shown in Table 5.

Plant pests usefully treated by the present invention include the downymildews. Non-limiting examples of such are shown in Table 2.

TABLE 1 Pests - Bacteria Disease Causative Agent Bacterial leaf blightPseudomonas avenae subsp. avenae and stalk rot Bacterial leaf spotXanthomonas campestris pv. holcicola Bacterial stalk rot Enterobacterdissolvens = Erwinia dissolvens Bacterial stalk and Erwinia carotovorasubsp. carotovora, Erwinia top rot chrysanthemi pv. Zeae Bacterialstripe Pseudomonas andropogonis Chocolate spot Pseudomonas syringae pv.Coronafaciens Goss's bacterial wilt Clavibacter michiganensis subsp.nebraskensis = and blight(leaf Corynebacterium michiganense pv.Nebraskense freckles and wilt) Holcus spot Pseudomonas syringae pv.Syringae Purple leaf sheath Hemiparasitic bacteria + (See under Fungi)Seed rot-seedling Bacillus subtilis blight Stewart's disease Pantoeastewartii = Erwinia stewartii (bacterial wilt) Corn stunt (Mesaachapparramiento, stunt, Spiroplasma kunkelii Central or Rio Grandestunt)

TABLE 2 Pests - Downy Mildews Disease Causative Agent Brown stripe downySclerophthora rayssiae var. zeae mildew Crazy top downy mildewSclerophthora macrospora = S. macrospore Green ear downy mildewSclerospora graminicola Java downy mildew Peronosclerospora maydis =Sclerospora maydis Philippine downy mildew Peronosclerosporaphilippinensis = Sclerospora philippinensis Sorghum downy mildewPeronosclerospora sorghi = Sclerospora sorghi Spontaneum downy mildewPeronosclerospora spontanea = Sclerospora spontanea Sugarcane downymildew Peronosclerospora sacchari = Sclerospora sacchari Dry ear rot(cob, kernel and Nigrospora oryzae (teleomorph: Khuskia oryzae) stalkrot) Ear rots, minor Aspergillus glaucus, A. niger, Aspergillus spp.,Cunninghamella sp., Curvularia pallescens, Doratomyces stemonitis =Cephalotrichum stemonitis, Fusarium culmorum, Gonatobotrys simplex,Pithomyces maydicus, Rhizopus microsporus, R. stolonifer = R. nigricans,Scopulariopsis brumptii Ergot (horse's tooth, diente Claviceps gigantea(anamorph: Sphacelia sp.) del caballo) Eyespot Aureobasidium zeae =Kabatiella zeae Fusarium ear and stalk rot Fusarium subglutinans = F.moniliforme var. subglutinans Fusarium kernel, root and Fusariummoniliforme teleomorph: Gibberella stalk rot, seed rot and fujikuroi)seedling blight Fusarium stalk rot, seedling Fusarium avenaceum(teleomorph: Gibberella root rot avenacea) Gibberella ear and stalk rotGibberella zeae (anamorph: Fusarium graminearum) Gray ear rotBotryosphaeria zeae = Physalospora zeae (anamorph: Macrophoma zeae) Grayleaf spot Cercospora leaf spot) Cercospora sorghi = C. sorghi var.maydis, C. zeae-maydis Helminthosporium root rot Exserohilumpedicellatum = Helminthosporium pedicellatum (teleomorph: SetosphaeriaHormodendrum ear rot (Cladosporium Cladosporium cladosporioides =Hormodendrum cladosporioides, rot), C. herbarum (teleomorph:Mycosphaerella tassiana) Hyalothyridium leaf spot Hyalothyridium maydisLate wilt Cephalosporium maydis Leaf spots, minor Alternaria alternata,Ascochyta maydis, A. tritici, A. zeicola, Bipolaris victoriae =Helminthosporium victoriae (teleomorph: Cochliobolus victoriae), C.sativus (anamorph: Bipolaris sorokiniana = H. sorokinianum = H.sativum), Epicoccum nigrum, Exserohilum prolatum = Drechslera prolata(teleomorph: Setosphaeria prolata) Graphium penicillioides,Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerella herpotricha,(anamorph: Scolecosporiella sp.), Paraphaeosphaeria michotii, Phoma sp.,Septoria zeae, S. zeicola, S. zeina Northern corn leaf blightExserohilum turcicum = Helminthosporium turcicum, Setosphaeria turcicaNorthern corn leaf spot Cochliobolus carbonum Helminthosporium ear rotBipolaris zeicola = Helminthosporium carbonum (race 1) Penicillium earrot (blue Penicillium spp., P. chrysogenum, P. expansum, eye, blue mold)P. oxalicum Phaeocytostroma stalk rot Phaeocytostroma ambiguum,Phaeocytosporella and root rot zeae Phaeosphaeria leaf spotPhaeosphaeria maydis, Sphaerulina maydis Physalospora ear rotBotryosphaeria Botryosphaeria festucae = Physalospora zeicola,(anamorph: Diplodia frumenti) Purple leaf sheath Hemiparasitic bacteriaand fungi Pyrenochaeta stalk rot and Phoma terrestris, Pyrenochaetaterrestris root rot Pythium root rot Pythium spp., P. arrhenomanes, P.graminicola Pythium stalk rot Pythium aphanidermatum = P. butleri L. Redkernel disease (ear Epicoccum nigrum mold, leaf and seed rot)Rhizoctonia ear rot Rhizoctonia zeae (teleomorph: Waitea circinata)Rhizoctonia root rot and Rhizoctonia solani, Rhizoctonia zeae stalk rotRoot rots, minor Alternaria alternata, Cercospora sorghi, Dictochaetafertilis, Fusarium acuminatum (teleomorph: Gibberella acuminata), F.equiseti (teleomorph: G. intricans), F. oxysporum, F. pallidoroseum, F.poae, F. roseum, F. cyanogena, (anamorph: F. sulphureum), Microdochiumbolleyi, Mucor sp., Periconia circinata, Phytophthora cactorum, P.drechsleri, P. nicotianae var. parasitica, Rhizopus arrhizus Rostratumleaf spot Setosphaeria rostrata, Helminthosporium (leaf disease, ear andstalk (anamorph: Exserohilum rostratum = rot) Helminthosporiumrostratum) Rust, common corn Puccinia sorghi Rust, southern cornPuccinia polysora Rust, tropical corn Physopella pallescens, P. zeae =Angiopsora zeae Sclerotium ear rot Sclerotium rolfsii (teleomorph:Athelia rolfsii) (southern blight) Seed rot-seedling blight Bipolarissorokiniana, B. zeicola = Helminthosporium carbonum, Diplodia maydis,Exserohilum pedicillatum, Exserohilum turcicum = Helminthosporiumturcicum, Fusarium avenaceum, F. culmorum, F. moniliforme, Gibberellazeae (anamorph: F. graminearum), Macrophomina phaseolina, Penicilliumspp., Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae,Sclerotium rolfsii, Spicaria sp. Selenophoma leaf spot Selenophoma sp.Sheath rot Gaeumannomyces graminis Shuck rot Myrothecium gramineumSilage mold Monascus purpureus, M. rubber Smut, common Ustilago zeae =U. maydis Smut, false Ustilaginoidea virens Smut, head Sphacelothecareiliana = Sporisorium holci-sorghi Southern corn leaf blightCochliobolus heterostrophus (anamorph: Bipolaris and stalk rot maydis =Helminthosporium maydis) Southern leaf spot Stenocarpella macrospora =Diplodia macrospora Stalk rots, minor Cercospora sorghi, Fusariumepisphaeria, F. merismoides, F. oxysporum, F. poae, F. roseum, F. solani(teleomorph: Nectria haematococca), F. tricinctum, Mariannaea elegans,Mucor sp., Rhopographus zeae, Spicaria sp. Storage rots Aspergillusspp., Penicillium spp. and other fungi Tar spot Phyllachora maydisTrichoderma ear rot and Trichoderma viride = T. lignorum teleomorph:root rot Hypocrea sp. White ear rot, root and stalk Stenocarpella maydis= Diplodia zeae rot Yellow leaf blight Ascochyta ischaemi, Phyllostictamaydis (teleomorph: Mycosphaerella zeae-maydis) Zonate leaf spotGloeccercospora sorghi

TABLE 3 Pests - Parasitic Nematodes Disease Pathogen Awl Dolichodorusspp., D. heterocephalus Bulb and stem (Europe) Ditylenchus dipsaciBurrowing Radopholus similes Cyst Heterodera avenae, H. zeae, Punctoderachalcoensis Dagger Xiphinema spp., X. americanum, X. Mediterraneum Falseroot-knot Nacobbus dorsalis Lance, Columbia Hoplolaimus Columbus LanceHoplolaimus spp., H. galeatus Lesion Pratylenchus spp., P. brachyurus,P. crenatus, P. hexincisus, P. neglectus, P. penetrans, P. scribneri, P.thornei, P. zeae Needle Longidorus spp., L. breviannulatus RingCriconemella spp., C. ornata Root-knot Meloidogyne spp., M. chitwoodi,M. incognita, M. javanica Spiral Helicotylenchus spp. Sting Belonolaimusspp., B. longicaudatus Stubby-root Paratrichodorus spp., P. christiei,P. minor, Quinisulcius acutus, Trichodorus spp. Stunt Tylenchorhynchusdubius

TABLE 4 Pests - Fungal Disease Fungal Pest Anthracnose leaf blight andColletotrichum graminicola anthracnose stalk rot (teleomorph: Glomerellagraminicola), Glomerella tucumanensis (anamorph: Glomerella falcatum)Aspergillus ear and kernel rot Aspergillus flavus Banded leaf and sheathspot Rhizoctonia solani = Rhizoctonia microsclerotia (teleomorph:Thanatephorus cucumeris) Black bundle disease Acremonium strictum =Cephalosporium acremonium Black kernel rot Lasiodiplodia theobromae =Botryodiplodia theobromae Borde blanco Marasmiellus sp. Brown spot(black spot, stalk rot) Physoderma maydis Cephalosporium kernel rotAcremonium strictum = Cephalosporium acremonium Charcoal rotMacrophomina phaseolina Corticium ear rot Thanatephorus cucumeris =Corticium sasakii Curvularia leaf spot Curvularia clavata, C.eragrostidis, = C. maculans (teleomorph: Cochliobolus eragrostidis),Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolusintermedius), Curvularia lunata (teleomorph: Cochliobolus lunatus),Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvulariasenegalensis, C. tuberculata (teleomorph: Cochliobolus tuberculatus)Didymella leaf spot Didymella exitalis Diplodia ear rot and stalk rotDiplodia frumenti (teleomorph: Botryosphaeria festucae) Diplodia earrot, stalk rot, seed Diplodia maydis = Stenocarpella rot and seedlingblight maydis Diplodia leaf spot or leaf streak Stenocarpella macrospora= Diplodia macrospore

TABLE 5 Pests - Rusts Host plant Common disease name Rust Species BarleyCrown rust Puccinia coronate Leaf rust Puccinia hordei Stem rustPuccinia graminis Stripe (yellow) rust Puccinia striiformis Corn Commonrust Puccinia sorghi Southern rust Puccinia polysora Tropical rustPhysopella pallescens, P. zeae = Angiopsora zeae Oats Crown rustPuccinia coronata Stem Rust Puccinia graminis Rye Stem rust Pucciniagramminis. = P. graminis f. sp. secalis Leaf (brown) rust Pucciniarecondita (anamorph: Aecidium clematidis) Sorghum Rust Puccinia purpureaSugarcane Common Rust Puccinia melanocephala. = P. erianthi Orange RustPuccinia kuehnii Wheat Leaf (brown) rust Puccinia triticina. = P.Recondita f. Sp. tritici = P. tritici-duri Stem (black) rust Pucciniagramminis = P. graminis f. sp. tritici Stripe (yellow) rust Pucciniastriiformis (anamorph: P. uredoglumarum) Apple American hawthorne rustGymnosporangium globosum Cedar apple rust Gymnosporangiumjuniperi-virginianae Japanese apple rust Gymnosporangium yamadae miyabeex yamada Pacific Coast pear rust Gymnosporangium libocedri Quince rustGymnosporangium clavipes Asparagus Rust Puccinia asparagi Banana Leafrust Uredo musae, uromyces musae Bean Rust Uromyces appendiculatus BeetSeedling rust. Puccinia subnitens Carrot Rust Aecidium foeniculiuromycesgraminis, Uromyces lineolatus subsp. nearcticus Chickpea Rust Uromycesciceris-arietini, Uromyces striatus Coffee Rust (orange or leaf rust)Hemileia vastatrix Rust (powdery or grey Hemileia coffeicola rust)Cotton Cotton rust Puccinia schedonnardi Southwestern rust Pucciniacacabata Tropical rust Phakopsora gossypii Eucalyptus rust Pucciniapsidii Flax Rust Melampsora lini Crape Rust Physopella ampelopsidisLettuce Rust Puccinia dioicae = P. extensicola var. hieraciata AsparagusAsparagus rust Puccinia asparagi Onion Onion rust Puccinia allii PeaRust Uromyces fabae peanut Rust Puccinia arachidis Pear Americanhawthome rust Gymnosporangium globosum Kern's pear rust Gymnosporangiumkernianum Pacific Coast pear rust Gymnosporangium libocedri Pear trellisrust Gymnosporangium fuscum Rocky Mountain pear rust Gymnosporangiumnelsonii Poplar European Poplar rust Melampsora larici-populinakleAmerican leaf rust Melampsora medusae Potato Common rust Pucciniapittierianap Deforming rust Aecidium cantensis Red clover Rust Uromycestrifolii-repentis Soybean Rust Phakopsora pachyrhizi Strawberry Leafrust Phragmidium potentillae = Frommea obtusa Sunflower Rust Pucciniahelianthi, P. xanthii, Uromyces junci Sweet potato Red rust Coleosporiumipomoeae

Pest Pathogenicity Genes

The skilled artisan can readily identify pest genes to target in thepresent invention. Such a gene could be any pest gene that serves adirect or indirect role in such a pest's deleterious effects on a hostplant. By way of example only, such a gene may be one that serves a rolein pest growth, development, replication and reproduction, and invasionor infection.

In one embodiment, the host plant does not contain a gene that is morethan about 70% homologous to the pest pathogenicity gene.

Optionally, the homology is not more than about 60%.

Optionally, the homology is not more than about 50%.

In one embodiment, the host plant does not contain a gene that containsa 25 nucleotide stretch that is more than about 70% homologous to thepest pathogenicity gene.

In one embodiment, the host plant does not contain a gene that containsa 25 nucleotide stretch that is more than about 60% homologous to thepest pathogenicity gene.

In one embodiment, the host plant does not contain a gene that containsa 25 nucleotide stretch that is more than about 50% homologous to thepest pathogenicity gene.

In one embodiment, the host plant does not contain more than one genethat contains a 25 nucleotide stretch that is more than about 70%homologous to the pest pathogenicity gene.

In one embodiment, the host plant does not contain more than one genethat contains a 25 nucleotide stretch that is more than about 60%homologous to the pest pathogenicity gene.

In one embodiment, the host plant does not contain more than one genethat contains a 25 nucleotide stretch that is more than about 50%homologous to the pest pathogenicity gene.

TABLE 6 Pests, Pest pathogenicity genes, and Host Plants Pest orpathogen group Pest pathogenicity gene Host Plants Fungi Cutinases AllKinases All Ribosomal RNAs All Adhesins All G-proteins All Elicitins AllBacteria Cutinases All Mascerating enzymes All Kinases All RibosomalRNAs All Adhesins All G-proteins All Insects Kinases All Ribosomal RNAsAll G-proteins All Moulting factors All Serine proteases All Cysteineproteases All Juvenile hormone esterase All Nematodes Kinases AllRibosomal RNAs All G-proteins All Cuticle collagen proteins AllCathepsin proteases All

By way of example, cutinase is a gene useful according to the presentinvention. When a fungal hypha invades a plant cell and absorbsnutrients, it absorbs the siRNAs from the plant and silences the fungus'essential and constitutive cutinase.

By way of example only, such a gene may be one that serves a role inpest growth, development, replication and reproduction, and invasion orinfection. Other non limiting examples include those in Table 6.

Sense (and Antisense) Sequences

Sense and antisense sequences according to the present invention can beany sequence with homology to a sense and antisense strand(respectively) of pest pathogenicity gene.

The sense and antisense sequences can be on the same DNA strand or ondifferent DNA strands. When the sense and antisense sequences are on thesame DNA strand, transcription of these sequences can be driven off thesame promoter or off of different promoters (e.g. different copies ofthe same promoter or different promoter). When the sense and antisensesequences are on different DNA strands, transcription of these sequencesis driven off of different promoters.

The sense and antisense sequences can be arranged in DNA ascomplementary, duplex DNA or the sequences can be in different regionsof the DNA (i.e. not forming duplex DNA with each other).

Each of the sense and antisense sequences comprise at least about 19nucleotides. Optionally, each sequence comprises at least about 50nucleotides, optionally at least about 100 nucleotides, optionally atleast about 150 nucleotides, optionally at least about 250polynucleotides, optionally at least about 500 nucleotides, optionallyat least about 600 polynucleotides.

In one embodiment, the sequences are modified from the pestpathogenicity gene to create or increase homology to the pathogenicitygene of more than one pest.

In another embodiment the sequences are modified from the pestpathogenicity gene to shorten an open reading frame.

In another embodiment the sequences are modified from the pestpathogenicity gene to result in less homology with a plant, animal, orhuman gene.

In another embodiment, sense and antisense sequences compriseduplicative regions of a pest pathogenicity gene. Selection of suchregions is made according to the teachings of the invention herein (e.g.highly conserved regions).

In other embodiments, regions of a pest pathogenicity gene or even theentire coding region of a pest pathogenicity gene may be duplicated tocomprise sense and antisense sequences to increase the length of thedouble stranded region formed by the sense and antisense sequences.While not bound by theory, it is believed that in some embodiments ofthe present invention, a threshold level of siRNA's must be formed toelicit useful gene silencing in the pest and to confer pest resistanceto the plant. Duplication of gene sequences is one useful way ofincreasing the length of the double stranded region and increasing thenumber of siRNA.

This (gene duplication) approach is demonstrated in examples hereinusing the heterologous polynucleotide pVZA300 (SEQ ID 7) comprising theelicitin INF1 of Phytophthora infestans (GenBank locus AY766228). Thissequence was selected because of its high homology to many otherelicitin genes expressed by the genus Phytophthora, suggesting that itcould silence many other elicitin genes and thereby provide resistanceto many species of Phytophthora. The sequence selected was 282 nt long,therefore the sequence was repeated (=564 nt) in both the sense andantisense orientations to approximate the lengths of the sense andantisense portions of pVZA100 (SEQ ID 5), pVZA200 (SEQ ID 6) and pVZA400(SEQ ID 8) that have been remarkably successful.

The success of this approach was demonstrated by the dramatic effect ofpVZA300 in reducing the growth and causing malformation of hyphae oftransformed Phytophthora nicotianae and Phytophthora sojae (FIG. 14 D,F) as compared to the effects of transformations with pCAMBIA1201,pVZA100, or pVZA200. The plasmids pVZA200 and pVZA300 are partiallyidentical with both containing a cathepsin gene, but pVZA300 alsocontains the repeated elicitin gene. Therefore, the deleterious impactof pVZA300 on the fungi must be due to the elecitin gene. This has verypositive implications for developing Phytophthora resistant plants andapplication of this approach to depathogenization of Phytophthoracontaminated soils.

Transforming Methods

A wide variety of methods are available for introducing heterologouspolynucleotides of the present invention into the target host underconditions that allow for stable maintenance and expression of thepolynucleotide. The particular choice of a transformation technologywill be determined by its efficiency to transform certain plant speciesas well as the experience and preference of the person practicing theinvention with a particular methodology of choice. It will be apparentto the skilled person that the particular choice of a transformationsystem to introduce nucleic acid into plant cells is not essential to ora limitation of the invention, nor is the choice of technique for plantregeneration.

While various transformation methods are taught herein as separatemethods, the skilled artisan will readily recognize that certain methodscan be used in combination to enhance the efficiency of thetransformation process. Non-limiting examples of such methods includebombardment with Agrobacterium coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

Direct delivery can be used to transform plant hosts according to thepresent invention. By way of non-limiting example, such direct deliverymethods include polyethylene glycol treatment, electroporation, liposomemediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29: 1353(1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228(1990d). One form of direct DNA delivery is direct gene transfer intoprotoplasts from, for example, embryogenic cell suspension cultures(Lazzeri and Lorz (1988) Advances in Cell Culture, Vol. 6, Academicpress, p. 291; OziasAkins and Lorz (1984) Trends in Biotechnology 2: 119).

Pollination Pathway

The skilled artisan is aware of certain challenges of genotype-dependanttransformation arising from low regeneration potential of cereals.Accordingly, in one embodiment of the present invention, transformationis accomplished by a genotype-independent transformation approach basedon the pollination pathway (Ohta Y., 1986). In maize, high efficiencygenetic transformation can be achieved by a mixture of pollen andexogenous DNA (Luo Z. X. and Wu R., 1988, Proc. Natl. Acad. Sci. USA83:715-719). Maize can be bred by both self-pollination andcross-pollination techniques. Maize has separate male and female flowerson the same plant, located on the tassel and the ear, respectively.Natural pollination occurs in maize when wind blows pollen from thetassels to the silks that protrude from the tops of the ears.

Transformation of rice according to the present invention can also beaccomplished via the pollen-tube pathway (Plant Molecular BiologyReporter 6:165-174). The major potential advantages of the pollen-tubepathway approach include: (a) genotype independence; (b) lack ofmosaicism; (c) no need for complicated cell and tissue culturetechniques.

Transformation of tomato and melon with heterologous polynucleotidesaccording to the present invention can be accomplished into intactplants via pollination pathway (Chesnokov, Yu. V., et al, 1992, USSRPatent No. 1708849; Bulletin of the USSR Patents, No. 4; Chesnokov Yu.V. & Korol A. B. 1993; Genetika USSR, 29:1345-1355). The procedures ofgenetic transformation based on the pollination-fecundation pathwayinclude: (i) employment of a mixture (paste) of the pollen andtransforming DNA; (ii) delivery of the alien DNA into the pollen tube,after pollination; and (iii) microparticle bombardment of microspores orpollen grains.

Agrobacterium Technology

In one embodiment of the present invention, host plants are transformedusing Agrobacterium technology. Agrobacterium-mediated transfer is awidely applicable system for introducing genes into plant cells becausethe DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. The useof Agrobacterium-mediated plant integrating vectors to introduce DNAinto plant cells is well known in the art. See, for example, the methodsdescribed by Fraley et al., (1985), Rogers et al., (1987) and U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety.

Agrobacterium-mediated transformation can efficiently be used withdicotyledonous host plants of the present invention including, by way ofnon-limiting example, Arabidopsis, corn, soybean, cotton, canola,tobacco, tomato, and potato.

Agrobacterium-mediated transformation is also applicable to nearly allmonocotyledonous plants of the present invention. By non-limitingexample, such monocotyledonous plant technologies are adaptable to rice(Hiei et al., 1997; Zhang et al., 1997; (Ishida et al., 1996); U.S. Pat.No. 5,591,616, specifically incorporated herein by reference in itsentirety), wheat (McCormac et al., 1998), and barley (Tingay et al.,1997; McCormac et al., 1998).

Agrobacterium-mediated transformation, according to the presentinvention, can be accomplished with cultured isolated protoplasts and bytransformation of intact cells or tissues.

Agrobacterium-mediated transformation in dicotyledons facilitates thedelivery of larger pieces of heterologous nucleic acid as compared withother transformation methods such as particle bombardment,electroporation, polyethylene glycol-mediated transformation methods,and the like. In addition, Agrobacterium-mediated transformation appearsto result in relatively few gene rearrangements and more typicallyresults in the integration of low numbers of gene copies into the plantchromosome.

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

When Agrobacterium (e.g. A. tumefaciens and A. rhizogenes) is used totransform plant cells according to the present invention, the DNA to beinserted can be cloned into special plasmids, namely either into anintermediate vector or into a binary vector. The intermediate vectorscan be integrated into the Ti or Ri plasmid by homologous recombinationowing to sequences that are homologous to sequences in the T-DNA. The Tior Ri plasmid also comprises the vir region necessary for the transferof the T-DNA. Intermediate vectors cannot replicate themselves inAgrobacteria. The intermediate vector can be transferred intoAgrobacterium tumefaciens by means of a helper plasmid (conjugation).

Binary vectors can replicate themselves both in E. coli and inAgrobacteria. Such vectors can comprise a selection marker gene and alinker or polylinker which are framed by the right and left T-DNA borderregions. They can be transformed directly into Agrobacteria (Holsters etal. [1978] Mol. Gen. Genet. 163:181-187). The Agrobacterium used as hostcell is to comprise a plasmid carrying a vir region. The vir region isnecessary for the transfer of the T-DNA into the plant cell. AdditionalT-DNA may be contained. The bacterium so transformed is used for thetransformation of plant cells. Plant explants can advantageously becultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenesfor the transfer of the DNA into the plant cell. Whole plants can thenbe regenerated from the infected plant material (for example, pieces ofleaf, segments of stalk, roots, but also protoplasts orsuspension-cultivated cells) in a suitable medium, which may containantibiotics or biocides for selection. The plants so obtained can thenbe tested for the presence of the inserted heterologous polynucleotides.No special demands are made of the plasmids in the case of injection andelectroporation. It is possible to use ordinary plasmids, such as, forexample, pUC derivatives.

Other transformation technology includes whiskers technology. See U.S.Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca.

Microprojectile Bombardment

Optionally, host plant cells are transformed in accordance with theinvention by microprojectile bombardment (U.S. Pat. No. 5,550,318; U.S.Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; and U.S. Pat. No.5,590,390; each of which is specifically incorporated herein byreference in its entirety). In this embodiment, particles are coatedwith nucleic acids and delivered into cells by a propelling force.Exemplary particles include those comprised of tungsten, platinum, andpreferably, gold. It is contemplated that in some instances DNAprecipitation onto metal particles would not be necessary for DNAdelivery to a recipient cell using microprojectile bombardment. However,it is contemplated that particles may contain DNA rather than be coatedwith DNA. Hence, it is proposed that DNA-coated particles may increasethe level of DNA delivery via particle bombardment but are not, in andof themselves, necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells are arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the microprojectilestopping plate.

An illustrative embodiment of a transforming method for deliveringsubject vectors into plant cells is by acceleration is the BiolisticsParticle Delivery System (BioRad, Hercules, Calif.), which is used topropel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered withplant cells cultured in suspension. The screen disperses the particlesso that they are not delivered to the recipient cells in largeaggregates. While not bound by theory, it is believed that a screenintervening between the projectile apparatus and the cells to bebombarded reduces the size of projectiles aggregate and may contributeto a higher frequency of transformation by reducing the damage inflictedon the recipient cells by projectiles that are too large.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themicroprojectile stopping plate. If desired, one or more screens may bepositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

One skilled in the art can optimize the prebombardment culturingconditions and the bombardment parameters to yield the maximum numbersof stable transformants. Both the physical and biological parameters forbombardment are important in this technology. Physical factors are thosethat involve manipulating the DNA/microprojectile precipitate or thosethat affect the flight and velocity of either the macro- ormicroprojectiles. Biological factors include all steps involved inmanipulation of cells before and immediately after bombardment, theosmotic adjustment of target cells to help alleviate the traumaassociated with bombardment, and also the nature of the transformingDNA, such as linearized DNA or intact supercoiled plasmids. It isbelieved that pre-bombardment manipulations are especially important forsuccessful transformation of immature embryos.

Accordingly, it is contemplated that the skilled artisan can adjust thebombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. Results from such small scale optimizationstudies are disclosed herein and the execution of other routineadjustments will be known to those of skill in the art in light of thepresent disclosure.

Transformation by microprojectile bombardment is widely applicable, andcan be used to transform virtually any plant species. Monocots areoptionally transformed by bombardment according to the presentinvention, for example maize (U.S. Pat. No. 5,590,390), barley (Ritalaet al., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety), rice(Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998),rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum(Casa et al., 1993; Hagio et al., 1991);

Dicots are optionally transformed by bombardment according to thepresent invention, for example, tobacco (Tomes et al., 1990; Buising andBenbow, 1994), soybean (U.S. Pat. No. 5,322,783, specificallyincorporated herein by reference in its entirety), sunflower (Knittel etal. 1994), peanut (Singsit et al., 1997), cotton (McCabe and Martinell,1993), tomato (Van Eck et al. 1995), and legumes in general (U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety).

Electroporation

In one embodiment, plant cells are transformed using the method ofelectroporation, See, for example, WO 87/06614 to Boyce ThompsonInstitute; U.S. Pat. Nos. 5,472,869 and 5,384,253, both to Dekalb; andWO 92/09696 and WO 93/21335, both to Plant Genetic Systems.

The method of Krzyzek et al. (U.S. Pat. No. 5,384,253, incorporatedherein by reference in its entirety), is well suited for the presentinvention. In this method, certain cell wall-degrading enzymes, such aspectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells. Alternatively, recipient cells are made moresusceptible to transformation by mechanical wounding.

The method of Tada, Y., et al (Efficient gene introduction to rice byelectroporation and analysis of transgenic plants: Use ofelectroporation buffer lacking chloride ions, Theor Appl Genet, Vol. 80:475-480, 1990) is, for example, suitable for transforming rice.

In one embodiment, to effect transformation by electroporation, friabletissues, such as a suspension culture of cells or embryogenic callus areused.

Optionally, immature embryos or other organized tissue are transformeddirectly. In this technique, cell walls are partially degraded byexposing them to pectin-degrading enzymes (pectolyases) or mechanicallywounding in a controlled manner.

Non-limiting examples plant cell hosts that can be transformed byelectroporation of intact cells according to the present invention arewheat (according to, for example, Zhou et al., 1993), tomato (accordingto, for example Hou and Lin, 1996), soybean (according to, for exampleChristou et al., 1987) and tobacco (Lee et al., 1989). See also U.S.Pat. No. 5,384,253; Rhodes et al., 1995; and D'Halluin et al., 1992

In one embodiment, plants are transformed for electroporation ofprotoplasts (according to, for example, Bates, 1994; Lazzeri, 1995).Non-limiting examples of plant cell hosts that can be transformed byelectroporation of protoplasts cells according to the present inventionare soybean plants (according to, for example, Dhir and Widholm in PCTPublication No. WO 92/17598, specifically incorporated herein byreference), barley (e.g., Lazerri, 1995), sorghum (e.g., Battraw et al.,1991), (e.g., Bhattacharjee et al., 1997), wheat (e.g., He et al., 1994)and tomato (e.g. Tsukada, 1989).

Viral Vectors

Furthermore, viral vectors can also be used to transform plantsaccording to the present invention. For example, monocotyledonous plantscan be transformed with a viral vector using the methods described inU.S. Pat. No. 5,569,597 to Mycogen Plant Science and Ciba-Giegy, nowNovartis.

U.S. Pat. No. 5,589,367 also describes plant transformation with a viralvector.

U.S. Pat. No. 5,316,931 describes plant transformation with a viralvector.

A large number of cloning vectors useful for higher plants (includingmonocots and dicots) according to the present invention comprise areplication system in E. coli and a marker that permits selection of thetransformed cells. These vectors comprise, for example, pBR322, pUCseries, M13 mp series, pACYC184, etc., and pCAMBIA 1201. Accordingly,the sequence comprising a heterologous polynucleotide of the presentinvention can be inserted into the vector at a suitable restrictionsite. The resulting plasmid is used for transformation into E. coli. TheE. coli cells are cultivated in a suitable nutrient medium, thenharvested and lysed. The plasmid is recovered. Sequence analysis,restriction analysis, electrophoresis, and other biochemical-molecularbiological methods are generally carried out as methods of analysis.After each manipulation, the DNA sequence used can be cleaved and joinedto the next DNA sequence. Each plasmid sequence can be cloned in thesame or other plasmids. Depending on the method of inserting desiredgenes into the plant, other DNA sequences may be necessary. If, forexample, the Ti or Ri plasmid is used for the transformation of theplant cell, then at least the right border, but often the right and theleft border of the Ti or Ri plasmid T-DNA, are joined as the flankingregion of the genes to be inserted (FIG. 1).

The use of T-DNA for the transformation of plant cells is described inEP 120 516; Hoekema (1985) In: The Binary Plant Vector System,Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et al.,Crit. Rev. Plant Sci. 4:1-46; An et al. (1985) EMBO J. 4:277-287; andmany subsequent references widely known and available to those skilledin the art.

Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ one or more marker genes into a heterologouspolynucleotide of the present invention.

Marker genes, according to the present invention, include those thatencode a selectable marker and those that encode a screenable marker,depending on whether the marker confers a trait that one can “select”for by chemical means, i.e., through the use of a selective agent (e.g.,a herbicide, antibiotic, or the like), or whether it is simply a“reporter” trait that one can identify through observation or testing,i.e., by “screening”. Of course many examples of suitable marker genesor reporter genes are known to the art and can be employed in thepractice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes that encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers that encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes that can be detectedby their catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA, and proteins that are inserted or trapped in the cell wall (e.g.,proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). The use of theHPRG (Stiefel et al., 1990) is well characterized in terms of molecularbiology, expression, and protein structure. However, any one of avariety of extensins and/or glycine-rich wall proteins (Keller et al.,1989) could be modified by the addition of an antigenic site to create ascreenable marker.

Elements of the present disclosure are exemplified in detail through theuse of particular marker genes. However in light of this disclosure,numerous other possible selectable and/or screenable marker genes willbe apparent to those of skill in the art in addition to the one setforth herein below. Therefore, it will be understood that the followingdiscussion is exemplary rather than exhaustive.

Selectable Markers

Selectable markers for use in connection with the present inventioninclude, but are not limited to, a neo gene (Potrykus et al., 1985) thatcodes for kanamycin resistance and can be selected for using kanamycin,G418, and the like; a bar gene that codes for bialaphos resistance; agene that encodes an altered EPSP synthase protein (Hinchee et al.,1988) thus conferring glyphosate resistance; a nitrilase gene such asbxn from Klebsiella ozaenae that confers resistance to bromoxynil(Stalker et al., 1988); a mutant acetolactate synthase gene (ALS) oracetohydroxyacid synthase gene (AHAS) that confers resistance toimidazolinone, sulfonylurea, or other ALS-inhibiting chemicals (EuropeanPatent Application 154,204); a methotrexate-resistant DHFR gene (Thilletet al., 1988); a dalapon dehalogenase gene that confers resistance tothe herbicide dalapon (U.S. Pat. No. 5,780,708); or a mutatedanthranilate synthase gene that confers resistance to 5-methyltryptophan (PCT Publication No. WO 97/26366). Where a mutant EPSPsynthase gene is employed, additional benefit may be realized throughthe incorporation of a suitable chloroplast transit peptide, CTP (U.S.Pat. No. 4,940,835). See also, Lundquist et al., U.S. Pat. No.5,508,468.

An illustrative embodiment of a selectable marker gene capable of beingused in embodiments of the present invention to select transformants arethose that encode the enzyme phosphinothricin acetyltransferase, such asthe bar gene from Streptomyces hygroscopicus or the pat gene fromStreptomyces viridochromogenes (U.S. Pat. No. 5,550,318, which isincorporated by reference herein). The enzyme phosphinothricin acetyltransferase (PAT) inactivates the active ingredient in the herbicidebialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase,(Murakami et al., 1986; Twell et al., 1989) causing rapid accumulationof ammonia and cell death.

Suitable selectable marker genes contemplated herein include anaminoglycoside phosphotransferase gene, neomycin phosphotransferasegene, G418 resistance gene, glyphosate resistance gene, hygromycinresistance gene, methotrexate resistance gene, imidazolinones resistancegene, sulfonylureas resistance gene, triazolopyrimidine herbicideresistance gene, ampicillin resistance gene, tetracycline resistancegene, bacterial kanamycin resistance gene, phosphinothricin resistancegene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene,amongst others.

Screenable Markers

Screenable markers that may be employed include, but are not limited to,a beta-glucuronidase or uidA gene (GUS) that encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., 1988); a beta-lactamase gene(Sutcliffe, 1978), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylegene (Zukowsky et al., 1983), which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an alpha-amylase gene (Ikuta et al.,1990); a tyrosinase gene (Katz et al., 1983), which encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone which in turncondenses to form the easily detectable compound melanin; abeta-galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), whichallows for bioluminescence detection; or even an aequorin gene (Prasheret al., 1985), which may be employed in calcium-sensitivebioluminescence detection, or a green fluorescent protein gene (Niedz etal., 1995).

Genes from the R gene complex are contemplated as useful screenablemarkers. The R gene complex encodes a protein that acts to regulate theproduction of anthocyanin pigments in most seed and plant tissue. Maizelines can have one, or as many as four, R alleles that combine toregulate pigmentation in a developmental and tissue specific manner.Thus, an R gene introduced into such cells will cause the expression ofa red pigment and, if stably incorporated, can be visually scored as ared sector. If a line carries dominant alleles for genes encoding theenzymatic intermediates in the anthocyanin biosynthetic pathway (C2, A1,A2, Bz1 and Bz2), but carries a recessive allele at the R locus,transformation of any cell from that line with R will result in redpigment formation. Exemplary lines include Wisconsin 22 that containsthe rg-Stadler allele and TR112, a K55 derivative that is r-g, b, P1.Alternatively any genotype of maize can be utilized if the C1 and Ralleles are introduced together.

It is further contemplated that R gene regulatory regions may beemployed in heterologous polynucleotides of the present invention inorder to provide mechanisms for controlling the expression of suchheterologous polynucleotides. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe et al., 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression ofgenes, e.g., insect resistance, drought resistance, herbicide toleranceor other protein coding regions. For the purposes of the presentinvention, it is believed that any of the various R gene family membersmay be successfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bol3). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening.

Marker Gene Strandedness

According to the present invention, marker genes can be designed byselection of sequence and position within the heteropolynucleotide toresult in a positive or negative marker. When a heteropolynucleotidecontains sequences of a marker gene but does not contain sufficientcomplementary sequences to the marker gene, a transcript of theheteropolynucleotide will contain the marker gene in a single strandedregion and it can be expressed. Thus expression is correlated withintegration of the heteropolynucleotide into the host plant. When aheteropolynucleotide contains sequences of a marker gene and sufficientcomplementary sequences to the marker gene, a transcript of theheteropolynucleotide will contain the marker gene in a double strandedregion and it will be silenced. Thus, expression of such a marker genewill indicate that the heteropolynucleotide is not being silenced in thehost plant (i.e. “negative marker”). When a plant cell is transformedwith a heteropolynucleotide comprising both a positive marker and anegative marker gene expresses the positive marker gene but does notexpress the negative marker gene, this indicates that the plant istranscribing and silencing at least part of the heteropolynucleotide.Thus expression is correlated with integration of theheteropolynucleotide into the host plant (i.e. positive sequences can beselected).

Choice of Marker Gene Sequence.

A skilled artisan will now readily recognize how combinations of markersat different positions within a heterologous polynucleotide of thepresent invention can be effected. Such differences in position effectwhether the marker is silenced or not (e.g. a gene within the regionthat forms a double stranded structure by way of hydrogen bonding isrecognized as foreign by the plant's internal defense mechanisms andhydrolyzed into siRNA [i.e. “silenced”]).

For example, in one embodiment, a heterologous polynucleotide of thepresent invention contains the hypothetical A gene (that is, any markergene of the present invention) upstream of the double stranded regionand a hypothetical B gene within the double stranded region. Anon-transformed cell is deficient in A expression (“A−). It is also B−and C−. A transformed cell that functionally inactivates B region (e.g.by gene silencing of the region wherein the B gene lies) is A+ and B−. Atransformed cell that fails to inactivate the B region is A+ and B+.

A skilled artisan will now recognize that the presence of antisensesequences of a marker gene, appropriately placed downstream of the sensesequences of the marker gene, will further facilitate gene silencing(e.g. double strand formation) of such marker gene.

It should also now be readily recognized that a marker localizeddownstream of a double stranded region will also be silenced if nopromoter lies between such double stranded region and such marker.

In one embodiment, heterologous polynucleotides of the present inventioninclude those with the structures set forth in Table 7.

TABLE 7 Heterologous polynucleotides

Promoter and Regulatory Elements

According to the present invention, a heterologous polynucleotide, or aportion thereof, is capable of being transcribed in a plant. In plants,such transcription requires a promoter that is operably linked to theheterologous polynucleotide. Such a promoter can be endogenous to theplant. By way of example, a heterologous polynucleotide can be insertedadjacent to a constitutive or inducible plant promoter.

The heterologous polypeptide of the present invention can optionallycontain a promoter. Such a promoter can be a plant promoter or anon-plant promoter that functions in a plant.

Plant promoters include but are not limited to ribulose-1,6-bisphosphate(RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter,beta-phaseolin promoter, ADH promoter, heat-shock promoters, and tissuespecific promoters such as a constitutive promoterwhere it is desirableto directing continuous gene expression in all cell types and at alltimes (e.g., actin, ubiquitin, CaMV 35S, and the like).

Promoters from a variety of sources can be used efficiently in plantcells to express foreign genes. For example, promoter regulatoryelements of bacterial origin, such as the octopine synthase promoter,the nopaline synthase promoter, the mannopine synthase promoter;promoters of viral origin, such as the cauliflower mosaic virus (35S and19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No.6,166,302, especially Example 7E) and the like may be used.

It may be desirable to use an inducible promoter, which is responsiblefor expression of genes in response to a specific signal, such as:physical stimulus (heat shock genes), light (RUBP carboxylase), hormone(Em), metabolites, chemical, and stress. Other desirable transcriptionand translation elements that function in plants may be used. Numerousplant-specific gene transfer vectors are known in the art.

It may be desirable to use a promoter that is active during a certainstage of the plant's development as well as active in plant tissues andorgans. Examples of such include but are not limited to pollen-specific,embryo-specific, corn-silk-specific, cotton-fiber-specific,root-specific, seed-endosperm-specific promoters and the like. Tissuespecific promoter regulatory elements can be used where it is desirableto promote transcription in tissues such as leaves or seeds (e.g., zein,oleosin, napin, ACP, globulin and the like).

While any promoter that functions in a plant cell (i.e. supportstranscription of a heterologous polynucleotide) is useful in the presentinvention, it can be desirable to select a relatively stronger or weakerpromoter. One embodiment of the present invention uses a strong promoterto drive the sense sequence of a pest pathogenicity gene and a weakerpromoter to drive the antisense sequence of the pest pathogenicity gene.One skilled in the art can readily determine promoter strengthempirically within the embodiments of the present invention. When anembodiment contains two promoters (driving two different transcripts),the relative transcription rate can be determined by quantifying thetranscripts (e.g. quantitative PCR).

Examples of promoters that are relatively strong are the figwort mosaicvirus promoter and the enhanced CaMV 35S promoter. An example of apromoter that is relatively weaker is the CaMV 35S promoter. One skilledin the art will readily appreciate that “weaker” and “stronger”promoters is a relative term, and they can be empirically identified by,for example, quantitative PCR with the appropriate primers against thetranscript that is driven by the promoters.

Other elements such as matrix attachment regions, scaffold attachmentregions, introns, enhancers, polyadenylation sequences and the like maybe present and thus may improve the transcription efficiency or DNAintegration. Such elements may or may not be necessary for DNA function,although they can provide better expression or functioning of the DNA.Such elements may be included in the DNA as desired to obtain optimalperformance of the transformed DNA in the plant. Typical elementsinclude but are not limited to Adh-intron 1, Adh-intron 6, the alfalfamosaic virus coat protein leader sequence, the maize streak virus coatprotein leader sequence, as well as others available to a skilledartisan.

Terminator

Heterologous polynucleotides of the present invention can optionallycontain a terminator.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Eukaryotic terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of poly (A)sequences to the 3′-end of a primary transcript.

Terminators active in cells derived from viruses, yeast, molds,bacteria, insects, birds, mammals and plants are known and described inthe literature and useful in the present invention. They may be isolatedfrom bacteria, fungi, viruses, animals and/or plants.

Examples of terminators suitable for use in the present inventioninclude the nopaline synthase (NOS) gene terminator of Agrobacteriumtumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35Sgene, the zein gene terminator from Zea mays, the Rubisco small subunit(SSU) gene terminator sequences, Subclover stunt virus (SCSV) genesequence terminators (International Patent Application No.PCT/AU95/00552), and the terminator of the Flaveria bidents malic enzymegene NSEA3 (PCT/AU95/00552).

Those skilled in the art will be aware of additional promoter sequencesand terminator sequences suitable for use in performing the invention.Such sequences may readily be used without any undue experimentation.

Embodiments of the present invention are taught herein where it isdesirable to have more than one terminator. Examples of such areembodiments are where the sense and antisense sequences are to becontained on separate transcripts (i.e. each having its own 3′ and 5′end).

Plant Regeneration and Propagation

Following transformation, a plant may be regenerated, e.g., from singlecells, callus tissue, or leaf discs, as is standard in the art. Almostany plant can be entirely regenerated from cells, tissues, and organs ofthe plant. Available techniques are reviewed in Vasil et al. (1984) inCell Culture and Somatic Cell Genetics of Plants, Vols. I, II, and III,Laboratory Procedures and Their Applications (Academic press); andWeissbach et al. (1989) Methods for Plant Molecular Biology, AcademicPress, 1989.

During suspension culture development, small cell aggregates (10-100cells) are formed, apparently from larger cell clusters, giving theculture a dispersed appearance. Upon plating these cells to solid media,somatic embryo development can be induced, and these embryos can bematured, germinated and grown into fertile seed-bearing plants.Alternatively, callus cells growing on solid culture medium can beinduced to form somatic embryos from which fertile seed bearing plantsmay develop. The characteristics of embryogenicity, regenerability, andplant fertility are gradually lost as a function of time in suspensionculture. Cryopreservation of suspension cells arrests development of theculture and prevents loss of these characteristics during thecryopreservation period.

The transformed plants may then be grown, and either pollinated with thesame transformed strain or different strains, and the resulting linehaving expression of the desired phenotypic characteristic identified.Two or more generations may be grown to ensure that expression of thedesired phenotypic characteristic is stably maintained and inherited,and then seeds harvested to ensure expression of the desired phenotypiccharacteristic has been achieved.

In addition to a plant, the present invention provides any clone of sucha plant, seed, selfed or hybrid descendants, and any part of any ofthese, such as cuttings, pollen, or seed. The invention provides anyplant propagule, that is any part which may be used in reproduction orpropagation, sexual or asexual, including cuttings, seed, and so on.Also encompassed by the invention is a plant which is a sexually orasexually propagated off-spring, clone, or descendant of such a plant;or any part or propagule of said plant, off-spring, clone, ordescendant. Plant extracts and derivatives are also provided.

Gene Stacking

A heterologous polynucleotide according to the present invention cancomprise a plurality of sense sequences and a plurality of antisensesequences (i.e. a “polycistronic heterologous polynucleotide”). Thus, aheterologous polynucleotide can protect a plant against multiple pests.

One skilled in the art will now recognize that, by using gene stacking,a plant can be made resistant to a plurality of pests. The plurality ofpests can be from the same phylum or from different phyla. For example,a plant can be made resistant to a plurality of fungal pests. As onenon-limiting example of gene stacking according to the presentinvention, soybeans can be made resistant to brown stem rot (Phialophoragregata), stem and root rot (Phytophthora sojae), white mold disease(Sclerotinia sclerotiorum), sudden death syndrome (Fusarium solanipisi), and Asian rust (Phakopsora pachyrhizi).

Optionally, a plant can be made resistant to a plurality of insectpests. As one non-limiting example of gene stacking according to thepresent invention, potatoes can be made resistant to potato leafhopper(Empoasca filament), Colorado potato beetle (Leptinotarsa decemlineata),and green peach aphid (Myzus persicae).

Optionally, a plant can be made resistant to a plurality of fungal andnon-fungal pests. As one non-limiting example of gene stacking accordingto the present invention, potatoes can be made resistant to potatogolden nematode (Globodera rostochiensis), Colorado potato beetle(Leptinotarsa decemlineata), and potato late blight (Phytophthorainfestans).

In one embodiment of the present invention, the heterologouspolynucleotide comprises two different sense and two different antisensesequences.

In one embodiment of the present invention, the heterologouspolynucleotide comprises at least three different sense and at leastthree different antisense sequences.

Plant pests often cause the infected plants to become yellow or brown,to be reduced in size, to produce less root exudates and to mature morerapidly. Therefore, controlling a single major pest of a plant may inturn make that plant more susceptible to other pests. If a fungaldisease of a plant is controlled, then the healthy plant will remainlarge and green for a longer period, and therefore be attractive to andsusceptible to insects and airborne fungal diseases. The green plantalso may produce more root exudates which will attract more nematodesand root infecting fungi. It has been discovered herein that it can besurprisingly advantageous to transform a plant with a heterologouspolynucleotide containing sequences homologous to a plurality of pestsgenes, wherein such genes are from pests of different taxons (e.g.subspecies, species, genera, families, orders, class).

Depathogenesis

It has been discovered that when a pest infects a host plant celltransformed according to the present invention, the pest becomes lesspathogenic to the transformed host plant cell and non-transformed hostplant cells. Moreover, in one embodiment, a pest infects a host plantcell transformed according to the present invention and then the genesilencing molecules derived from the plant by feeding are subsequentlytransmitted through mating (e.g. by cytoplasmic transmission,conjugation, hyphal anastomosis, etc.) to populations of the pest andproduce a less pathogenic pest. In this way, populations of pests can bemade non-pathogenic (i.e. less pathogenic) by, for example, a pest thatis unable to multiply, and the macroenvironment (e.g. soil, plantfoliage, plant debris, buildings) contaminated with a pest can beprotected or decontaminated therefrom.

Optionally, progeny of the pest are less pathogenic to the transformedhost plant cell and non-transformed host plant cells.

Accordingly, one embodiment of this invention is a mechanism forbiological control, and it has great economic value in that it can beused to deplete important fields of pathogenic pest isolates of fungiand reduce the application of pesticides. In this embodiment thetransgenic plant transmits the siRNAs to the pest by way of pest feedingbehavior, and the pest loses its pathogenicity due to the subsequentsilencing of its own pathogenic gene. Moreover, the pest progeny will inturn be nonpathogenic (e.g. by cytoplasmic transmission, mating,conjugation, hyphal anastomosis), thereby effectively cleaning up thelocal environment of that pest and preventing or significantly reducingits plant destroying behavior.

Depathogenesis, in one embodiment, is accomplished by co-cultivatingnonpathogenic (either transformed directly or having obtained siRNAs),pests with pathogenic pests. Such co-cultivation is accomplished, by wayof nonlimiting example, by broadcast of the transgenic pest to grow andinfest the soil wherein the nonpathogenic pests then mate with likespecies. Such broadcast can be accomplished for fungi, by way ofnonlimiting example, by cultivating transformed fungi on barley or wheatseeds and then distributing such seeds to soil that is in need of beingdepathogenized or protection from pathogenic pests.

This embodiment is suitable for protecting established valuable plantssuch as trees (e.g. redwoods, oaks, palms, maples, etc.).

Although the foregoing discussion uses fungal plant pathogens as anexample, the strategy for depathogenesis can be adapted to any otherplant pests that would transmit the “silenced” trait to their progeny,as will be readily apparent to the skilled artisan.

First, it is possible to “depathogenize” a pest by silencing a pestpathogenicity gene. The pest then could survive in the environment (e.g.soil) and spread the siRNAs throughout the population and no longerattack plants. Second, by silencing major structural genes or essentialnutrition genes such as ribosomal RNA or elicitin genes, respectively,it is possible to debilitate or kill a pest thereby eliminating it fromcrop fields or lowering its presence below economic thresholds.

EXAMPLES Example 1 Cloning Strategy

The plasmids pVZA100, pVZA200, pVZA300, and pVZA400 are embodiments thatwere designed for transformation of various plant species, including,but not limited to, tobacco (Nicotiana tobacum, cv. Xanthi), soybean(Glycine max cv. Williams 82), potato (Solanum tuberosm cv. Alpha) andcorn (Zea mays cv. Hi II×B73). pVZA100 contains the full-length cutinasegene from Phytophthora nicotianae in both the sense (S) and antisense(AS) orientations, separated by a spacer region containing a portion(817 bp) of the E. coli β-glucuronidase gene (GUS) referred to as dGUS.pVZA100 expression in plants is under the control of the cauliflowermosaic virus (CaMV) 35S promoter and the cucumber mosaic virus (CMV)capsid protein gene 5′ untranslated region (5′ UTR). The terminationsignal also is from the CaMV 35S gene. The backbone of the pVZA100plasmid is the plasmid pCAMBIA1201 (FIG. 1) (Roberts et al., 2000). Itsplant selectable marker is the antibiotic hygromycin. The expression ofhygromycin phosphotransferase in pCAMBIA 1201 also is controlled by theCaMV 35S promoter and terminator. The reporter gene in pCAMBIA 1201 isGUS with a catalase intron, also with the CaMV 35S promoter, but withthe nopaline synthase (NOS) terminator.

Example 2 Construction of the Intermediary Plasmid pVZA1

Because of its versatility, the gene expression plasmid pUC18 cpexp(Slightom, 1991) was selected to contain the gene silencing cassette.This plasmid is pUC18, containing the CaMV 35S promoter, followed by theCMV capsid protein gene 5′ UTR and the CaMV 35S gene termination signal.A Bgl II site was engineered into the polylinker between the CMV 5′ UTRand the CaMV 35S terminator. To obtain dGUS, the plasmid pCAMBIA1201(FIG. 1) was double digested with Nco I and Hinc II. The GUS fragment821 bp in length, corresponding to nucleotides 9540 to 10361 ofpCAMBIA1201, was isolated and purified by agarose gel electrophoresis.That fragment was then digested (blunted) at the 3′ Nco I site usingmung bean nuclease to reduce it to 817 bp. This blunt-ended dGUS wasthen re-purified by agarose gel electrophoresis and ligated into the BglII site of pUC18 cpexp to yield the intermediary plasmid pVZA1 (FIG. 2).

Example 3 Construction of the pVZA2, pVZA3, pVZA100, pVZA200, pVZA300and pVZA400 Plasmids

The full length cutinase gene from P. nicotianae was amplified in theantisense orientation by PCR from a cutinase clone in the plasmidpZErO-2.1 (Munoz and Bailey, 1998) using primers VZA 1F (SEQ ID NO: 1)and VZA 2R (SEQ ID NO: 2) containing the Not I and Nco I restrictionsites, respectively. It was then purified by agarose gelelectrophoresis, and ligated into the Not I and Nco I sites of theplasmid pVZA1. The resulting plasmid, pVZA2, was cloned in E. coli andpurified. A copy of the cutinase gene in the sense orientation wasamplified by PCR from the same cutinase clone in the plasmid pZErO-2.1using primers VZA 3F and VZA 4R (SEQ ID NO: 3 and SEQ ID NO: 4,respectively) containing the Apa I and Xho I restriction sites,respectively. It was purified by agarose gel electrophoresis, andligated into the Apa I and Xho I restriction sites in the plasmid pVZA2to produce the final intermediary plasmid pVZA3 (FIG. 3). The portioncontaining dGUS and separating the cutinase gene in sense and antisenseorientation is referred to as the silencing construct. The portioncontaining the silencing construct plus the regulatory elements for theconstruct (35S promoter, the 5′ UTR and the 35S terminator) is referredto as the silencing cassette (FIG. 3).

The silencing cassette was excised from plasmid pVZA3 by doublerestriction digestion with EcoR I and Hind III and purified by agarosegel electrophoresis. The plasmid pCAMBIA1201 was double digested withEcoR I and Hind III, and the silencing cassette was ligated into thosesites to produce the final plant transformation plasmid pVZA100. Thepresence of the entire silencing cassette was confirmed by restrictiondigestion of pVZA100 with PstI (which releases the whole insert) and bydigestions with the various restriction enzymes used in its assembly.The digestion products were separated by agarose gel electrophoresis andcompared with known molecular standards. The sequence, 2201 nt, of thesilencing construct was confirmed by sequencing of the DNA, and it isSEQ ID NO: 5. The following oligonucleotide pairs were used as PCRprimers to detect the presence of the respective genes in pVZA100 and intransgenic plants: cutinase (SEQ ID NOs: 1 and 2, or SEQ ID NOs: 3 and4); GUS (SEQ ID NOs: 9 and 10) and hygromycin phosphotrasferase (SEQ IDNOs: 11 and 12).

Plant transformation plasmids pVZA200, pVZA300 and pVZA400 were based onthe same principle as pVZA100. The silencing construct was an invertedrepeat of the selected pest pathogenicity gene in both the sense andantisense orientations, separated by the dGUS fragment. However, thesilencing constructs for pVZA200, pVZA300, and pVZA400 were synthesizedcommercially by GenScript Corporation (Piscataway, N.J.) with ApaI andNot I restriction sites at the 5′ and 3′ ends respectively, and clonedin the pUC57 vector.

The silencing construct of pVZA200 contained nucleotides 221 to 820 ofthe cathepsin B gene (GenBank locus AY702822) of the aphid Myzuspersicae. This aphid is a major pest and the most efficient vector ofmany plant viruses. Cathepsin B is a cysteine protease, a digestiveenzyme required for the survival and development of Myzus persicae. The2031 nt sequence of the silencing construct for pVZA200 is shown as SEQID NO: 6.

The regulatory elements were then added to the pVZA200 silencingconstruct to yield the pVZA200 silencing cassette. For this, the entirepVZA100 silencing cassette was excised from pVZA100 by digestion withEcoR I and Hind III and purified by agarose gel electrophoresis. It wascloned into a similarly digested pUC19. This plasmid was then digestedwith ApaI and Not I. The pUC19 portion containing the regulatoryelements from pVZA100 was purified and ligated to the pVZA200 silencingconstruct released from pUC57 by digestion with ApaI and Not I. ThepVZA200 silencing cassette, now flanked by the regulatory elements frompVZA100, was increased in pUC19. The entire pVZA200 silencing cassettewas excised from pUC19 by digestion with EcoR I and Hind III and thenligated into the EcoR I and Hind III sites of pCAMBIA1201 to completethe pVZA200 plant transformation plasmid. As with pVZA100, the presenceof the entire silencing cassette was confirmed by restriction digestionof pVZA200 with PstI and by digestions with the various restrictionenzymes used in its assembly. The digestion products were separated byagarose gel electrophoresis and compared with known molecular standards.

The following oligonucleotide pairs were used as PCR primers to detectthe presence of the respective genes in pVZA200 and in transgenicplants: cathepsin (SEQ ID NOs: 38 and 39); GUS (SEQ ID NOs: 9 and 10)and hygromycin phosphotrasferase (SEQ ID NOs: 11 and 12).

The plant transformation plasmids pVZA300 and pVZA400 were plannedsimilarly and synthesized. The silencing construct of pVZA300 was adouble gene construct, containing gene sequences from two differentplant pests. It was designed to contain the same cathepsin B geneinverted repeat around the dGUS fragment as the silencing construct ofpVZA200. But in addition, at each end it contained the partial codingsequence for the elicitin INF1 of Phytophthora infestans (GenBank locusAY766228). This elicitin gene is required by Phytophthora infestans forpathogenicity and as a sterol receptor because Phytophthora spp. do notsynthesize the sterols essential for their growth and development. Theelicitin sequence was 282 nt long. Therefore, to obtain a sequenceapproaching 600 nt, the sequence was repeated to obtain 564 nt.

An ApaI restriction site was included at the 5′ end of the elicitinsense sequence, and a NotI restriction site was included at the 3′ endof the elicitin antisense sequence. The elicitin sense sequence wasadded to the 5′ end of the pVZA200 sequence, and the elicitin antisensesequence was added to the 5′ end of the pVZA200 sequence to complete thesilencing construct of pVZA300. It is 3159 nt long and composed of theelicitin gene and cathepsin gene both in the sense orientation, followedby dGUS, followed by the cathepsin gene and elicitin gene, respectively,both in the antisense orientation.

The sequence of the silencing construct for pVZA300 is shown as SEQ IDNO: 7. This sequence was synthesized by GenScript Corporation and clonedin the pUC57 vector. The regulatory elements were then added asdescribed for pVZA200 to complete the silencing cassette for pVZA300,and it was cloned into the EcoR I and Hind III sites of pCAMBIA1201. Aspreviously, the presence of the entire silencing cassette was confirmedby restriction digestion of pVZA300 with PstI and by digestions with thevarious restriction enzymes used in its assembly. The digestion productswere separated by agarose gel electrophoresis and compared with knownmolecular standards. The following oligonucleotide pairs were used asPCR primers to detect the presence of the respective genes in pVZA300and in transgenic plants: elicitin and cathepsin overlap (SEQ ID NOs: 40and 39) cathepsin (SEQ ID NOs: 38 and 39); GUS (SEQ ID NOs: 9 and 10)and hygromycin phosphotrasferase (SEQ ID NOs: 11 and 12).

The silencing construct of the plant transformation plasmid pVZA400contained nt 1-600 of the ribosomal RNA genes (rDNA) of Phytophthorainfestans (GenBank locus AJ854293) in the sense and antisenseorientations, respectively, separated by dGUS. An ApaI and NotIrestriction site were included on the 5′ and 3′ ends, respectively. Thesequence of the silencing construct (2031 nt) for pVZA400 is shown asSEQ ID NO: 8. It was synthesized by GenScript Corporation and cloned inthe pUC57 vector. The regulatory elements were then added as describedfor pVZA200 to complete the silencing cassette for pVZA400, and it wascloned into the EcoR I and Hind III sites of pCAMBIA1201. As previously,the presence of the entire silencing cassette was confirmed byrestriction digestion of pVZA400 with PstI and by digestions with thevarious restriction enzymes used in its assembly. The digestion productswere separated by agarose gel electrophoresis and compared with knownmolecular standards.

The following oligonucleotide pairs were used as PCR primers to detectthe presence of the respective genes in pVZA400 and in transgenicplants: ribosomal DNA (SEQ ID NOs: 41 and 42); GUS (SEQ ID NOs: 9 and10) and hygromycin phosphotrasferase (SEQ ID NOs: 11 and 12).

Example 4 Transformation Methods

Plant cells (including tobacco, soybean, potato and corn) weretransformed with transformation plasmids pCAMBIA1201, pVZA100, pVZA200,pVZA300, and pVZA400 using biolistics or with pVZA100 using strain PGV2260 of Agrobacterium tumefaciens. Fungal cultures were transformed onlyby biolistics with transformation plasmids pCAMBIA1201, pVZA100,pVZA200, pVZA300, and pVZA400.

Preparation of tungsten or gold microprojectiles for bombardment withthe helium-driven biolistic device was according to the protocol ofShark et al. 1991. The microprojectiles were coated with eitherpCAMBIA1201, pVZA100, pVZA200, pVZA300, pVZA400, or with no DNA. Forplant tissue and fungal culture transformation, the Dupont PDS-1000apparatus was used under vacuum (23 mm Hg) and at 1200 PSI with tissuesat 8 and 12 cm from the stopping plate. For seedling and plantlettransformation, a hand held homemade particle gun was used at 200 PSI,and held as close as possible to the developing meristem.

The transformations with Agrobacterium tumefaciens were performedessentially as described by Rogers et al. (1987). Although biolisticsand Agrobacterium tumefaciens transformation are used to exemplify thisportion of the subject invention, any method of introducing heterologousDNA into target cells for purposes of transformation that is known inthe art could be used, as would readily be appreciated by the ordinarilyskilled artisan, and as would be optimized depending on the targetspecies.

Example 5 Tobacco Transformation with pVZA100 and Regeneration

Organogenic callus cultures of Nicotiana tabacum cv Xanthi weremaintained in shake cultures in the laboratory and transferred monthlyto fresh MS medium (Murashige and Skoog, 1962). Calli were removed fromflasks aseptically, filtered free of liquid and spread on sterile filterpaper in a sterile plastic Petri dish. They were bombarded with pVZA100according to the methods described above, and the filter paper wastransferred to phytohormone-free MS medium overnight. The following daythe filter paper was transferred to MS medium containing 1 mg/16-benzyladenine, 0.1 mg/l napthaline acetic acid, 1 mg/l thiamine-HCl,100 mg/l inositol, 30 g/l sucrose, 6 g/l phytagar and 30 ug/mlhygromycin for selection of the transgenic cells. For regeneration, theexpanding calli were transferred to fresh MS medium every 2 weeks.Emerging shoots were then transferred to MS medium without phytohormonesto form roots. Rooted plantlets were transferred to soil or testeddirectly for resistance to P. nicotianae. Plants were grown to maturityin soil and seed collected for subsequent analysis.

Example 6 Fungal Transformation and Selection Protocols

Five mm mycelial discs of Phytophthora nicotianae, P. sojae and P.infestans were bombarded according to the methods and with theconstructs described in Examples 3 and 5. Bombarded cultures were placedon plates of V-8 agar and incubated at 27 C overnight. The following daythey were transferred to V-8 agar plates containing 400 ug/ml hygromycinand incubated at 27 C for 3-4 days. Discs (5 mm) were cut from the newgrowth and transferred to Petri dishes containing 15 ml of V-8 broth andincubated at 27 C for 2 days. The mycelial mats were washed with sterilewater, pressed dry and incubated for 2 days with 10 ml of soil extractto induce sporangia formation. To induce zoospore release, the mats wereplaced in sterile demineralized water and chilled at 4 C for 30-60 min.Zoospores were collected by centrifugation for 5 min. at 5,000 g and 4C. For selection, the zoospore concentration was adjusted toapproximately 1,000/ml and plated on V-8 agar containing 400 ug/mlhygromycin and X-gluc (Jefferson et al., 1986) and incubated for severaldays at 27 C.

Those isolates stably transformed with the GUS gene in pCAMBIA1201appeared on the X-gluc agar as small blue colonies originating fromsingle zoospores. They were transferred to individual plates of V-8 agarcontaining 400 ug/ml hygromycin, and the cultures consistently stainedblue, positive for GUS, after many transfers. Those isolates transformedwith the pVZA100, pVZA200, pVZA300, and pVZA400 silencing plasmids alsoinitially appeared as small blue colonies originating from singlezoospores. They were transferred to plates of V-8 agar containing 400ug/ml hygromycin and X-gluc. After 2-3 transfers on this medium, thosecolonies which survived became white, indicating that the GUS gene hadbeen silenced. All cultures were maintained at 27 C and transferredmonthly to fresh medium.

Example 7 Transformation of Corn, Soybeans and Potato

Corn was transformed by bombarding Type II callus from Hi II germplasmwith gold particles coated with two plasmids, pVZA100 and pBAR184 (Frameet al., 2000). The pBAR184 was included so that the co-transformantscould be selected and regenerated on media containing the herbicidebialaphos rather than hygromycin. Calli were grown and selected onN6-based media containing 2 mg/l each of 2,4-dichlorophenoxyacetic acidand bialaphos. For regeneration and rooting, embryos were transferred toMS media containing 2 mg/l of bialaphos, and no phytohormones. Rootedplantlets were assayed by PCR.

Soybean was transformed both by Agrobacterium tumefaciens andbiolistics. Mature embryos were excised aseptically from hydrated seedsof Williams soybean. The embryos were then soaked for 15 min. in a fewmilliters of a culture of Agrobacterium tumefaciens that had been grownon a shaker overnight at 28 C and contained either pCAMBIA1201 orpVZA100. The treated embryos were placed on MS medium containing 15 mg/Lof hygromycin and phytohormones (BAP and NAA) and then on MS medium freeof phytohormones for rooting. Embryo-like structures formed andgerminated, and leaves were produced (FIG. 6), but no plants wereregenerated and rooted, despite testing of many media. Biolistictransformation of soybean was performed using the hand held homemadeparticle gun. Seedlings 8-10 days old and 6-10 cm tall were bombardedwith gold particles coated with either pCAMBIA1201, pVZA100, pVZA200,pVZA300 or pVZA400 or with no DNA. The plants were grown in thegreenhouse and assayed by PCR. Mature seed was collected and tested forresistance to P. sojae.

Potato was transformed both by Agrobacterium tumefaciens and biolistics.Russett Burbank and Alpha potatoes were transformed by Agrobacteriumtumefaciens. Tubers were surface disinfested, and the potato “eyes” wereexcised and plated on solid MS medium without phytohormones. The eyesgerminated, and plantlets were produced by cutting a node sectioncontaining one leaf and placing the stem in fresh MS medium. Rootedplantlets 5-7 cm tall were produced in 10-15 days. The leaves wereremoved from the plantlets, and the stems were cut into pieces 1 cmlong. The pieces were then soaked as above for 15 min. in a fewmilliters of a culture of Agrobacterium tumefaciens that had been grownon a shaker overnight at 28 C and contained either pCAMBIA1201 orpVZA100. The pieces were blotted on sterile filter paper and the tissueculture procedure of Cearley and Bolyard (1997) was followed. Bulboustuber-like and root-like structures were produced (FIG. 8), but noplants were regenerated and rooted, despite testing of many media. Forbiolistic transformation, potato plantlets 3-5 cm tall were transplantedto soil. After 2-3 days they were bombarded using the hand held homemadeparticle gun and gold particles coated with either pCAMBIA1201, pVZA100,pVZA200, pVZA300 or pVZA400 or with no DNA. The plants were grown in thegreenhouse and assayed by PCR and for their reactions to aphids and P.infestans.

Example 8 DNA and RNA Extraction from Plant and Fungal Tissues and PCRand RT/PCR Protocols

Total DNA was extracted from tobacco leaf tissue and mycelium ofPhytophthora nicotianae by the method of Dellaporta et al., 1983.Usually a 100 mg sample of frozen tissue was ground to a fine powder ina microfuge tube containing liquid nitrogen, and then homogenized in 500ul of extraction buffer with a motor-driven stainless steel pestle.

Total RNA for RT/PCR reactions was extracted from leaf tissue and fungalmycelium by the TRIzol method of Invitrogen. Usually 100 mg of tissuewas frozen in liquid nitrogen in a 1.8 ml microfuge tube. It was groundto fine powder and then homogenized in 1.0 ml of TRIzol with amotor-driven pestle. The mixture was allowed to incubate for 5 min. atroom temperature. Then 200 ul of chloroform were added and mixed byvortexing. The suspension was incubated 5 min. at room temperature. Thetubes were centrifuged at 14,000 rpm for 15 min. at 4 C. The aqueousphase was transferred to a clean tube, 500 ul of isopropanol were addedand the samples mixed and incubated 10 min. at room temperature. Thetubes were centrifuged again for 10 min. at 4 C, and the supernatant wasdiscarded. The pellet was rinsed once with 250 ul of 75% ethanol byvortexing and centrifuging 5 min. at 4 C. The RNA pellet was air driedbriefly, dissolved in 20 ul of RNase-free water and stored at −70 Cuntil used.

A similar method was used for large preparations of total RNA for siRNAdetection. Usually 1 gram of frozen leaf or fungal mycelium was groundto fine powder in liquid nitrogen and homogenized with 15 ml TRIzolreagent. Then 3 ml of chloroform were added and incubated as above. Thesupernatant was recovered by centrifugation and the RNA precipitatedwith ½ volume of isopropanol. The pellet was washed with 75% ethanol,air dried and resuspended in 80 ul of RNase-free water.

The PCR reactions for both plant and fungal tissues were performedessentially as described by Munoz and Bailey, 1998, except the reactionvolumes totaled 50 ul. The reactions contained 1×PCR buffer (10×solution: 500 mM KCl, 100 mM Tris-HCl pH 9.0, and 1% Triton X-100), 2.5mM MgCl₂, 200 μM of each deoxyribonucleotide triphosphate (dNTP), 2.5units of Taq DNA polymerase (Invitrogen or Promega), 100 pmol of theappropriate forward and reverse oligonucleotide primer (see SEQ IDNOs:), 1-10 ul of template and sufficient deionized water to equal 50ul. DNA preparations used as templates were usually diluted to 50 ng permicroliter, and 1 microliter was used as the template.

The PCR reaction conditions were standardized except for the annealingtemperature, which was varied depending on the Tm of the oligonucleotideprimers for the gene being amplified. The standardized conditions were94 C for 2 min. for melting, followed by 40 cycles of 1 min. at 94 C, 1min. at the annealing temperature, and 2 min. at 72 C for elongation.After 40 cycles there was 10 min. elongation at 72 C, and the reactionswere held at 4 C until assayed.

The RT/PCR reactions were carried out in 3 separate steps, DNasetreatment, reverse transcription and then PCR, as recommended byInvitrogen. The DNAse treatment was performed to eliminate any DNAcarried over in the RNA purification to ensure that only messenger RNAwas being transcribed, and not the genomic DNA. The DNase mix containedper reaction: 3 ug of RNA, 1 ul 10×DNAse I buffer, 2 ul DNAse I, 0.5 ulRNase OUT and water (RNase-free) to 10 ul final volume. This wasincubated at 26 C for 30 min. Then 6.5 ul of 25 mM EDTA were added pertube and incubated for 15 min. at 65 C.

For the reverse transcriptase (RT) reactions a Mix I and Mix II wereprepared. Mix I contained per reaction: 1 ul dNTPs, 4 ul 5×RT buffer,and 2 ul dithiothreitol. Mix II contained per reaction: 0.5 ul RNaseOUT, 1 ul reverse transcriptase and 1 ul water. The appropriateoligonucleotide primers (1 ul each or water) were placed in theappropriate tubes and Mix I and II added appropriately. Mix I was placedin every tube, Mix II in the odd numbered tubes and 2.5 ul water in theeven numbered tubes; then 10 ul of DNase treated RNA was added to eachtube. The RT reactions were incubated at 42 C for 60 min, and then at 70C for 15 min.

Example 9 Detection of siRNAs in Plant and Fungal Tissues

The procedure was essentially as described by Hutvagner et al., 2000.Total RNA was extracted from tobacco leaves or fungal mycelium as above.It was quantitated in a spectrophotometer at 260 nm and 80 ug werefractionated by denaturing polyacrylamide gel electrophoresis on 15%gels for 2.5 hrs at 0.025 Amp. The gels were stained with ethidiumbromide, photographed, and the RNA transferred to hybond N+ (Amersham)for 1 hr. at 10 volts at 4 C. Hybridization was performed as describedby Sambrook et al. (1989) for Northern blotting.

The probe was transcribed from a 500 by PCR product of the cutinase geneand labeled with ³²P by random priming. Membranes were prehybridized 1hr at 50 C with HYBAID buffer solution from Amersham and hybridized forat least 16 hrs at 50 C. Washes were performed twice with 5×SSC at roomtemperature. The hybridization signals were detected by phosphorimaging,using a Storm 860 phosphorimager scanner from Molecular Dynamics.

Example 10 GUS Staining of Plant and Fungal Tissues

Preparation of the X-GLUC substrate: For 200 ml, set up a beaker with150 ml distilled water and add the following components: Sodiumphosphate buffer pH 7.0 (1 M), 20 ml, EDTA pH 8.0 (500 mM), 4 ml,Potassium ferrocyanide K4Fe(CN)6.3H2O, 0.042 g, Triton X-100 0.2 ml.Stir for 10 min. Adjust pH 7.0 with NaOH. Dissolve 100 mg of X-GLUC in 2ml DMSO or dimethyl-formamide. Add the X-GLUC in DMSO to the beaker.Filter sterilize the solution, dispense in 10 ml aliquots, and store at−20 C. For incorporation into growth media, add 100 mg of X-GLUC powderdissolved in DMSO to 250 ml V8 agar cooled and ready to dispense forfungal isolates or to MS media for plant materials.

Staining procedure: Add enough X-GLUC solution to cover the smallexplant of plant tissue and incubate at 37 C for 3-24 hours. For fungalisolates or plant materials in X-gluc agar plates, incubate at 24-27 Cfor 3-24 hours.

This procedure is essentially as described by Jefferson, et al., 1986.

Example 11 Plant Inoculation Tests with Fungi

Three methods were tested to inoculate tobacco with P. nicotianae. Allgave satisfactory results, but the toothpick method was more rigorousand more dependable.

1. Toothpick method: This method was provided by Dr. A. Csinos (personalcommunication). Round wooden toothpicks were broken in half andautoclaved in V-8 broth. The sterile toothpicks were laid flat in platesof V-8 agar, and 6-8 mycelial discs (5 mm in diameter) of P. nicotianaewere spaced equidistant on the plate. The plates were incubated at 27 Cfor at least 2 weeks, when chlamydospores become obvious by microscopicexamination. Plant inoculation is accomplished by standing 1 or 2toothpicks in the soil next to the crown of the plant. The plants werecovered with a plastic tent to maintain relative humidity at 100% andincubated at 27 C. Symptoms were usually apparent after 2-4 days, andsusceptible plants were usually dead after 5-8 days. The reactions ofresistant transgenic plants vary from being symptomless to producingrestricted stem lesions 1-4 cm long, which do not interfere with plantgrowth and subsequent flower and seed development.

2. The mycelial soak method: Mycelia of P. nicotianae were grownovernight at 27 C in V-8 broth. Transgenic seedlings or plants in soilwere washed free of the medium and placed in 100 ml of sterile deionizedwater. Mycelial mats were shredded with forceps and added to the water.The plants were covered with a plastic tent to maintain relativehumidity at 100% and incubated at 27 C. Symptoms were usually apparentafter 2-4 days, and susceptible plants were usually dead after 5-8 days.The roots and crown of the susceptible plants were completely macerated(FIG. 11A). The roots of the resistant transgenic plants appear to be“burned” at the tips (FIG. 11B), or there were defined lesions at thecrown, but this does not interfere with plant growth and subsequentflower and seed development after the plants were potted in soil.

3. Zoospore inoculation: Abundant sporangia were produced by placingstrips of surface-sterilized tobacco leaves in growing cultures of P.nicotianae at 27 C in V-8 broth overnight. Millions of zoospores werereleased, by placing the mats in sterile demineralized water and chilledat 4 C for 30-60 min. Zoospores were collected by centrifugation for 5min. at 5,000 g and 4 C.

Soybean plants were inoculated with P. sojae by two different methods.Seedlings were inoculated by cutting a 3 mm cylinder from an activelygrowing culture on V8 agar with a cork borer and placing the cylinder inthe axil of one hypocotyl. The inoculum was held in place by Parafilm,and the plants enclosed in a plastic bag to give 100% humidity. Leavesof soybean plants were inoculated with P. sojae by placing a leaf in aPetri dish with 10 ml of sterile water and adding 1 or 2 plugs ofinoculum as above.

Potato leaves were inoculated with P. infestans by placing a leaf in aPetri dish with 10 ml of sterile water and adding 1 or 2 plugs ofinoculum as above, or by placing 10 ul of a zoospore suspension perleaflet.

In all inoculation tests, the plant or leaf reactions were read andrecorded daily until the inoculated plants or leaves stopped dying orshowed no evidence of infection.

Example 12 Molecular Characterization of P. nicotianae Resistant TobaccoPlants

DNA and RNA were extracted from T0 and T2 generation transformed plantsof Example 27. Polymerase chain reaction (PCR) and reversetranscription/PCR(RT/PCR) tests were performed on the DNA and RNAsamples, respectively. The PCR reactions were performed to determine thepresence of the transgenes in the plants. The RT/PCR reactions wereperformed to determine if the transgenes were being expressed or hadbeen silenced. The results were the same for both the T0 and T2generations, and are summarized in Table 8.

TABLE 8 Gene Detection and Expression in Transgenic and Wild TypeTobacco Plants Hygromycin phosphotransferase GUS Tissue Assayed PCRRT-PCR PCR RT-PCR Tobacco wild type − − − − Tobacco +pCAMBIA1201 + + + + pVZA100 Transgenic line 4 + + + − pVZA100 Transgenicline 23 + + + − pVZA100 Transgenic line 26 + + + − pVZA100 Transgenicline 27 + + + − pVZA100 plasmid DNA + na + na 16S Ribosomal Cutinase RNATissue Assayed PCR RT-PCR PCR RT-PCR Tobacco wild type − − + + Tobacco +pCAMBIA1201 − − + + pVZA100 Transgenic line 4 + − + + pVZA100 Transgenicline 23 + − + + pVZA100 Transgenic line 26 + − + + pVZA100 Transgenicline 27 + − + + pVZA100 plasmid DNA + na − na + = Positive (gel band) −= Negative (no gel band) na = Not applicable

In the upper panel of Table 8, the data in the first column demonstratethat the hygromycin resistance gene (hygromycin phosphotransferase) wasnot present in the wild type tobacco plants, but it was present in allthe plants transformed with either the pCAMBIA 1201 plasmid alone orwith pVZA100. The data in the second column demonstrate that thehygromycin phosphotransferase gene was expressed in all plants in whichit was present. The data in the third column demonstrate that the GUSgene was not present in the wild type tobacco plants, but it was presentin all the plants transformed with either the pCAMBIA 1201 plasmid aloneor with pVZA100. The data in the fourth column demonstrate that the GUSgene was expressed only in the pCAMBIA 1201 transformed plants, where noGUS silencing construct was present. However, GUS was silenced in alltransgenic plants containing the pVZA100 silencing construct.

In the lower panel of Table 8, the data in the first column demonstratethat the fungal cutinase gene was not present in the wild type tobaccoplants, nor in those plants transformed with the pCAMBIA 1201 plasmidalone. However, it was present in all the plants transformed withpVZA100. The data in the second column demonstrate that the fungalcutinase gene was not expressed in either the wild type plants or inthose plants transformed with either the pCAMBIA 1201 plasmid alone orwith pVZA100. The data in the third column demonstrate that the 16Sribosomal RNA gene was present in the wild type and all of thetransgenic tobacco plants, but not in the silencing construct. The datain the fourth column demonstrate that the 16S ribosomal RNA gene wasexpressed in all of the tobacco plants, whether wild type or transgenic.

When the wild type tobacco plants or those transformed with pCAMBIA1201were inoculated with P. nicotianae, they were always susceptible,whereas those transformed with pVZA100, such as Transgenic lines 4, 23,26 and 27 were resistant (FIGS. 11A and B, respectively).

Example 13 Fungal Transformation and Characterization

Different cultures of either P. nicotianae or P. sojae are bombardedwith either pCAMBIA1201, pVZA100, pVZA200, pVZA300, or pVZA400 as above,and single zoospore cultures are produced from the viable cultures.

Those P. nicotianae cultures bombarded with pCAMBIA1201 and pVZA100 weretested for pathogenicity on the normally susceptible wild type Xanthitobacco. Those cultures transformed with pCAMBIA1201 remainedpathogenic, whereas those cultures transformed with pVZA100 werenonpathogenic. Wild type and transgenic cultures of P. nicotianae weregrown in liquid V8 medium and DNA and RNA were extracted as describedabove. Those samples were analyzed by PCR and RT/PCR as above. Theresults are summarized in Table 9.

TABLE 9 Gene Detection and Expression in Transgenic and Wild TypePhytophthora nicotianae Hygromycin phosphotransferase GUS Tissue AssayedPCR RT-PCR PCR RT-PCR P. nicotianae wild type − − − − P. nicotianae +pCAMBIA 1201 + + + + P. nicotianae + pVZA100 + + + − pVZA100 plasmidDNA + na + na 16S Ribosomal Cutinase RNA Tissue Assayed PCR RT-PCR PCRRT-PCR P. nicotianae wild type + + + + P. nicotianae + pCAMBIA1201 + + + + P. nicotianae + pVZA100 + − + + pVZA100 plasmid DNA + na −na + = Positive (gel band) − = Negative (no gel band) na = Notapplicable

In the upper panel of Table 9, the data in the first column demonstratethat the hygromycin phosphotransferase gene was not present in the wildtype P. nicotianae, but it was present in those cultures of P.nicotianae transformed with either the pCAMBIA 1201 plasmid alone orwith pVZA100. The data in the second column demonstrate that thehygromycin phosphotransferase gene was expressed in all cultures inwhich it was present.

The data in the third column demonstrate that the GUS gene was notpresent in the wild type P. nicotianae, but it was present in thosecultures of P. nicotianae transformed with either the pCAMBIA 1201plasmid alone or with pVZA100. The data in the fourth column demonstratethat the GUS gene was being expressed only in the pCAMBIA 1201transformed P. nicotianae, where no GUS silencing construct was present.However, GUS was silenced in all transgenic P. nicotianae culturescontaining the pVZA100 silencing construct.

In the lower panel of Table 9, the data in the first column demonstratethat the fungal cutinase gene was present in the wild type P.nicotianae, as well as those cultures transformed with either thepCAMBIA 1201 plasmid alone or with the pVZA100 silencing construct,because cutinase is a constituitive gene of P. nicotianae. The data inthe second column demonstrate that the fungal cutinase gene wasexpressed in both the wild type P. nicotianae as well as in the culturestransformed with the pCAMBIA 1201 plasmid. However, the cutinaseexpression was silenced in those P. nicotianae cultures transformed withthe pVZA100 silencing construct.

The data in the third column demonstrate that the 16S ribosomal RNA genewas present in the wild type and all of the transgenic P. nicotianae,but not in the silencing construct. The data in the fourth columndemonstrate that the 16S ribosomal RNA gene was being expressed in boththe wild type and transgenic P. nicotianae cultures.

Microscopic examination of cultures of P. nicotianae and P. sojaebombarded with pCAMBIA1201, pVZA100 and pVZA200 indicated that all grewnormally like the wild type and with no visual malformations (FIG. 14A). Similar results were observed for cultures of P. nicotianaereisolated from infected wild type tobacco or tobacco transformed withpVZA100. However, those cultures of P. nicotianae bombarded with pVZA300or pVZA400 (FIG. 14 B, C, D) and those of P. sojae also bombarded withpVZA300 or pVZA400 (FIG. 14 E, F) grew very slowly, their hyphae wereseverely malformed, and some cultures died.

Transformation of P. nicotianae and P. sojae with pCAMBIA1201 andpVZA200 had no deleterious effects on the fungi because those plasmidsdo not contain fungal genes and cannot induce silencing in those fungi.Furthermore, transformation of P. nicotianae and P. sojae with pVZA100,which contains the P. nicotianae cutinase gene, allowed them to grownormally, but caused both species to become nonpathogenic, because thecutinase gene, which is essential for pathogenicity, was silenced bypVZA100.

Transformation of P. nicotianae and P. sojae with pVZA300 or pVZA400,which contain the P. infestans elicitin and ribosomal RNA genes,respectively, both had marked deleterious effects on the growth anddevelopment of both P. nicotianae and P. sojae. There was insufficientgrowth even to provide enough inoculum for pathogenicity tests. Thissuggests that the elicitin and ribosomal RNA genes were silenced in thetransformed fungi, and that silencing either of these important genes isvery detrimental or lethal to the fungi. This is further supported bythe fact that both pVZA200 and pVZA300 contain the Myzus persicaecathepsin gene. But in addition, pVZA300 contains the P. infestanselicitin gene. Since pVZA200 caused no visible effects, and pVZA300caused detrimental effects, the elicitin gene appears to be responsiblefor the malformation caused by pVZA300.

As above, where P. nicotianae cutinase induced plant resistance to otherspecies of Phytophthora, these results demonstrate that essential genesfrom one species of Phytophthora (P. infestans), when introduced insilencing constructs, can cause very detrimental effects on otherspecies of Phytophthora (P. nicotianae and P. sojae), which should beuseful in controlling Phytophthora spp. directly in plants or bydepathogenesis of infested soil and fields.

Example 14 Demonstration of Gene Silencing in Tobacco by siRNA Detection

Further proof that gene silencing is occurring is the detection of thesmall interfering RNAs (siRNAs) in the purportedly silenced transformedplants (Examples 5 and 13) and transformed fungus (Example 13). ThesiRNAs are 21 to 25 nucleotides (nt) in length, with homology to themRNA that is being “silenced”.

Total RNA was extracted from wild type and purportedly silencedtransgenic T0 and T1 generation tobacco plants and wild type andpurportedly silenced P. nicotianae. The RNA was separated bypolyacrylamide gel electrophoresis, blotted to a membrane and hybridizedwith a probe transcribed from a 500 bp PCR product of the cutinase geneand labeled with ³²P by random priming. The hybridization and detectionconditions were as in Example 9 above.

FIGS. 4 a and 4 b show the results of hybridization of the cutinase geneprobe to RNAs from wild type and transgenic tobacco plants andtransgenic P. nicotianae. In FIG. 4 a, lane 1 shows hybridization to amixture of the 35 nt oligonucleotides VZA 3F (SEQ ID NO: 3) and VZA 4R(SEQ ID NO: 4). These were used to prime the PCR reaction to synthesizethe template for production of the probe. Therefore, they are completelyhomologous to the template, and also of a known molecular size. Lanes 2and 3 contain RNA from wild type tobacco plants. There is nohybridization because neither the cutinase gene nor the silencingconstruct are present in wild type plants, hence no siRNAs are produced.Lanes 4 to 11 contain the RNA from T0 and T1 generation plants oftransgenic lines 4, 23, 26 and 27. The hybridization demonstrates thepresence of the siRNAs in each of the transgenic plants, indicating thedegradation of the cutinase mRNA, and the concomitant silencing of thecutinase gene in the transgenic plants.

In FIG. 4 b, lane 1 again shows hybridization to a mixture of the 35 ntoligonucleotides VZA 3F and VZA 4R. Lane 2 shows hybridization to theRNA from the wild type P. nicotianae. The intense spot 620 nt in lengthcorresponds to the full length transcript of the cutinase gene in wildtype P. nicotianae. The cutinase gene is constituitive in this culture,and no silencing construct is present. Therefore, the full length mRNAtranscript survives and is not digested to siRNAs. Lanes 3 to 6 containthe RNA from four cultures of P. nicotianae transformed with thesilencing construct. The hybridization demonstrates the presence ofsiRNAs in each of the transgenic cultures of P. nicotianae, indicatingthe degradation of the cutinase mRNA, and the concomitant silencing ofthe cutinase gene in the transgenic P. nicotianae.

Biological evidence for the silencing of the essential cutinase gene intransgenic P. nicotianae comes from the fact that those culturestransformed with the cutinase gene silencing construct, pVZA100, arerendered non-pathogenic, whereas those cultures transformed with thepCAMBIA 1201 plasmid alone remain equally pathogenic to the wild typecultures from which they were derived. Further evidence of genesilencing in the presence of the pVZA100 silencing construct is providedby the GUS gene. The GUS reaction was always positive in those culturesthat were transformed with pCAMBIA1201 and were pathogenic. Whereas itwas always negative in those cultures that were transformed with pVZA100and were nonpathogenic. This correlates directly with the activity orsilencing of the cutinase gene in the same culture.

Example 15 Highly Homologous Phytophthora Cutinase Sequences UsefulHerein

Analysis of Phytophthora gene sequences provides a basis for designingheterologous polynucleotides that confer cross-species resistance. As anillustration of the use of bioinformatic analysis to select pestpathogenicity gene sequences useful according to the present invention,25 different isolates of Phytophthora were cloned and sequenced.Isolates included the nine species P. capsici, P. cinnamomi, P.citricola, P. citrophthora, P. hevea, P. megakarya, P. megasperma (P.sojae), P. nicotianae and P. palmivora. Each of these sequences (SeeSequence ID Nos 13 to 37) are useful for polynucleotides of the presentinvention.

For example, nine of the sequences are identical and 11 are >99%identical to the cutinase gene sequence of P. nicotianae. A shadedletter

indicates a difference in sequence.

TABLE 10 The nucleotide sequences of the cutinase genes of 25isolates comprising 9 species of Phytophthora. (VZA 1 65R = Pnic Ri)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCG

CGTAGTCAGCATCGGTGTA (SEQ ID NO: 13) (VZA 2 69F = Pnic 21)ACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACG (SEQ ID NO: 14) (VZA 3 73F = Pnic 23)

ACGAACGTCACGAACCC (SEQ ID NO: 15) (VZA 4 88F = Pnic 17)

CGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGGG (SEQ ID NO: 16)(VZA 5 62R = Pcap Ri)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCG (SEQ ID NO: 17)(VZA 6 70F = Pcap 30)CCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGT

ACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCT (SEQ ID NO: 18) (VZA 7 72F = Pcap 30)

TCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCCTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCT (SEQ ID NO: 19) (VZA 8 79F = Pcap 28)ACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCC (SEQ ID NO: 20) (VZA 9 84F =Pcap 01)

CTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGC

(VZA 10 87F = Pcap 32)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAG

(SEQ ID NO: 22)NO: 22) (VZA 11 89F = Pcap 03)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTCAGATTCACGTCGTT (SEQ ID NO: 23)(VZA 12 90F = Pcap 22)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGG (SEQ ID NO: 24)(VZA 13 91F = Pcap 25)

TCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGGG (SEQ ID NO: 25)(VZA 14 81F = Ppal 02)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGG (SEQ ID NO: 26)(VZA 15 82F = Ppal 10)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCG (SEQ ID NO: 27)(VZA 16 83F = Ppal 11)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTITCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAG

(SEQ ID NO: 28) (VZA17 85F = Ppal 04)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGG (SEQ ID NO: 29)(VZA 18 86F = Ppal 05)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGGG (SEQ ID NO: 30)(vza 19 64R = Pcin Ri)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCAT

CTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGATTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGG (SEQ ID NO: 31)(VZA 20 77F = Pcin 01)

CACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGG (SEQ ID NO: 32) (VXA 21 63R =Pcto Ri) TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCAT

CCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCGG (SEQ ID NO: 33)(VZA 22 66R = Pctr Ri)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCAT

TCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAGCGTAGTCAGCATCGGTGTAGCACG (SEQ ID NO: 34) (VZA 23 80F = Pmek 01)TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCATCGCGTCGGCGATCATGTCGTCGGTACCAACATGGCGAATCCGTAGCTGCTCGTCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCCATTGACGAACGTCACGAACCCGCTAACACTCTTTCGGTCATCAGGGTCATTAG

(SEQ ID NO: 35) (VZA 24 92F = Pmeg 01)

CTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGC

GTCAGCATCGGTGTAGCACGTCAGATTCACGTCGTTCCCG (SEQ ID NO: 36) (VZA 25 93F =Phev 02) TCATACCATCTCGACAATGCCTCGAACTTGTCCTTCGGCAACGGCTTCGTCAT

TCCTCCACTAGGTGGCGCACCAAGTGGAACTTGTTCATAATATGCTTGCTCTTGCTGTGCTTGCCAGGCTTGGCAGTTAGATAGATGCACGACATGTTATCGCCGAGTATCTCCGGAGTCGCAAACTCCCAGCATAGTTCGTCACAGAGTCCACGTAGCCACTGTAGATCTCTGGTACCTTCATTCATAGCAATATACTCTGCTTCCGTTGTGCTCTGTGCGTTGATCTCTTGCTTTCTTGATCCGTACGAAACCACATTGCC

CGTAGTCAGCATCGGTGTAGCACGTCAGATTCACGTCGT (SEQ ID NO: 37)

Without being bound by theory, these cutinase sequences provide themolecular basis for the cross resistance demonstrated above and supportthe general application of gene sequencing and bioinformatic analysis toselect other pest pathogenicity genes with similar outcomes.

Example 16 Intergeneric Resistance in Tobacco was Conferred by the P.nicotianae Cutinase Gene-Silencing to a Disease Caused by a Peronosporatabacina

A natural epidemic of the blue mold disease of tobacco caused byPeronospora tabacina occurred in the greenhouse containing the tobaccoplants. Clear differences in blue mold severity were observed. Threegenotypes of plants were available for evaluation: Transgenic plantscontaining the cutinase gene and which had already been selected forresistance to P. nicotianae, cutinase transgenic plants which had notyet been selected for resistance to P. nicotianae, and wild type ornon-transgenic Xanthi tobacco plants. The numbers in the photographs inFIG. 5 refer to the different disease ratings, not the different plantlines or genotypes.

TABLE 11 Reactions of Different Tobacco Genotypes to a Natural Epidemicof Blue Mold of Tobacco Caused by Peronospora tabacina in the GreenhouseDisease Rating and Analysis Tobacco Genotype 1* 2 3 4 #Plants 1 + 2#Plants 3 + 4 Phytophthora resistant 34** 11  9 12 45 (68)*** 21 (32)Unselected transgenic 62  20 67 26 82 (47) 93 (53) Wild Type Xanthi 0  3 12 30 3 (7) 42 (93) *= Disease ratings. **= Number of plants in thatclass. ***= Percentage of plants in that class. 1 = Symptomless 2 =Obvious symptoms 3 = Severe symptoms 4 = Dead or dying

Table 11 shows that among the P. nicotianae resistant plants, 68% ofthem were moderately affected by Peronospora tabacina (disease ratings1+2), and 32% were severely affected (disease ratings 3+4); about a 2:1ratio in favor of resistance to Peronospora tabacina. For the transgenicunselected plants, 47% were moderately affected and 53% severelyaffected; about a 1:1 ratio in favor of resistance to Peronosporatabacina. Of the wild type plants, 7% were moderately affected and 93%were severely affected; about a 13:1 ratio in favor of susceptibility.Therefore, FIG. 5 and Table 11 clearly demonstrate different levels ofresistance in the different tobacco genotypes, and that the cutinasegene from P. nicotianae induces broad based resistance in tobacco toPeronospora tabacina, a fungus distantly related to P. nicotianae.

Table 12 demonstrates that Phytophthora nicotianae and Peronosporatabacina are not closely related taxonomically. Yet, as shown above, thePhytophthora nicotianae cutinase gene confers resistance to bothPhytophthora nicotianae and Peronospora tabacina. Therefore, thePhytophthora nicotianae cutinase gene provides broad resistance againstat least three different fungal species and three fungal diseases. Itprotects against two different species of Phytophthora, P. nicotianae intobacco and P. sojae in soybeans, and it also protects tobacco againstboth Phytophthora nicotianae and Peronospora tabacina. These two fungalspecies are classified in the same order, but in distinctly differentfamilies and genera (Table 12).

TABLE 12 Taxonomic relationship between Phytophthora nicotianae andPeronospora tabacina Phytophthora nicotianae Peronospora tabacina ClassOomycetes Oomycetes Order Peronosporales Peronosporales FamilyPythiaceae Peronosporaceae Genus Phytophthora Peronospora SpeciesPhytophthora nicotianae Peronospora tabacina

Example 17 Soybeans Transformed and Made Resistant with PhytophthoraGene Silencing Constructs

Soybean tissue culture: Shoots emerged from many of the soybean embryostransformed with pVZA100 (Example 7) by A. tumefaciens and by biolistics(FIG. 6). Other calli on the plate remained green and became veryfriable (FIG. 7A). DNA was extracted from three samples each of theshoots and friable calli, and PCR was performed using theoligonucleotide primers designed to amplify the GUS (SEQ ID NOs: 9 and10) and hygromycin phosphotransferase (SEQ ID NOs: 11 and 12) genes.

Both the GUS and hygromycin phosphotransferase genes (HPT) were detectedin the soybean callus, as shown in FIG. 7B. The upper panel of FIG. 7Bshows that all 6 of the samples (lanes 1-6) were transgenic for HPT(lane 7 is the plasmid control). The lower panel shows that at least 4(lanes 1, 2, 4, 5) of the 6 samples were transgenic for the GUS gene(lane 7 is the plasmid control). These data demonstrate that the hostplant cells were transgenic with the pVZA100 heterologouspolynucleotide.

The continued growth of the plantlets and shoots on the mediumcontaining 15 mg/L of hygromycin demonstrate that the calli expressedthe heterologous polynucleotide.

The fact that the soybean calli were GUS negative in their stainingreaction demonstrated that GUS expression in the host plant cell wassilenced by the heterologous polynucleotide.

Next, the calli are grown, rooted, transplanted to soil, and grown tomaturity to obtain pollen and seed. Non transformed plants arepollinated with pollen from a mature transformed plant, and theresultant fertilized seed is grown to a mature plant. Mature plants (F0plants), plants grown from seeds of an F0 plant, and non-transformedplants regenerated from seeds from transformed plant pollen andnon-transformed plant flowers all show resistance to Phytophthora sojae.

Soybean plants: This example (17) with soybean tissue culture wasclearly demonstrated in Example 27 wherein soybean plants transformed bybombardment with pVZA100 were resistant to P. sojae (FIG. 13). Thisresult also demonstrates that broad resistance was conferred by thecutinase gene of P. nicotianae against P. sojae.

Example 18 Potatoes Transformed and Made Resistant to Phytophthorainfestans

From Example 7, both roots and minitubers were produced in the cultureplates (FIG. 8). The plantlets, roots, and minitubers grew on the mediumcontaining 15 mg/L of hygromycin demonstrating that the calli weretransformed and expressing the hygromycin phosphotransferase transgene.Potato calli transformed with pCAMBIA1201 were GUS positive staining,whereas potato calli transformed with pVZA100 were GUS negativestaining. This demonstrates that GUS was silenced in those tissuestransformed with pVZA100.

PCR analysis demonstrates the presence of the transgenes. Progeny plantsfrom cuttings and tubers test positive for siRNAs. The potatoestransformed with the silencing construct are resistant to Phytophthorainfestans.

Potato plants: This example (18) with potato tissue culture was clearlydemonstrated in Example 27 wherein potato plants transformed bybombardment with pVZA300 and pVZA400 were resistant to P. infestans.

Example 19 Soybeans are Made Resistant to Brown Stem Rot (Phialophoragregata rDNA)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Phialophora gregata ribosomal RNA gene (rDNA)(GenBank locus U66728, SEQ ID NO: 43). Soybean plants are generated fromtransformed cells and are successfully selected for conferred resistanceto brown stem rot following inoculation with Phialophora gregata. Plantsresistant to Phialophora gregata are also resistant to Phakopsorapachyrhizi and Aspergillus nidulans.

Example 20 Soybeans are Made Resistant to Brown Stem Rot (Phialophoragregata Genotype B DNA Marker)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Phialophora gregata ribosomal RNA gene (rDNA)(GenBank locus U66728, SEQ ID NO: 44). Soybean plants are generated fromtransformed cells and are successfully selected for conferred resistanceto brown stem rot following inoculation with Phialophora gregata. Plantsresistant to Phialophora gregata are also resistant to Phakopsorapachyrhizi and Aspergillus nidulans.

Example 21 Soybeans are Made Resistant to Stem and Root Rot(Phytophthora sojae Cutinase)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Phytophthora sojae cutinase (i.e. SEQ ID NO:36). Soybean plants are generated from transformed cells and aresuccessfully selected for conferred resistance to stem and root rotfollowing inoculation with Phytophthora sojae.

Example 22 Soybeans are Made Resistant to White Mold Disease(Sclerotinia sclerotiorum RNA Polymerase Subunit)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Sclerotinia sclerotiorum partial rpb2 gene forRNA polymerase II second largest subunit, exon 1, strain 484 (GenBanklocus AJ745716, SEQ ID NO: 45). Soybean plants are generated fromtransformed cells and are successfully selected for conferred resistanceto white mold disease following inoculation with Sclerotiniasclerotiorum. Plants resistant to Sclerotinia sclerotiorum are alsoresistant to Phakopsora, Aspergillus nidulans, Magnaporthae orizae,Candida sojae, Gibberella zeae, and Puccinia graminis. Transformedplants show normal growth, lacking a gene with high homology to the S.sclerotiorum rpb2 gene. Similar results are obtained using sense andantisense sequences corresponding to Puccinia hordei 18S ribosomal RNAgene, partial sequence of GenBank locus AY125412 (SEQ ID NO: 46).

Example 23 Soybeans are Made Resistant to White Mold Disease(Sclerotinia sclerotiorum Hexose Transporter)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Sclerotinia sclerotiorum hexose transporter(hxt2) gene (GenBank locus AY647268.1, SEQ ID NO: 47). Soybean plantsare regenerated from transformed cells and successfully selected forconferred resistance to white mold disease following inoculation withSclerotinia sclerotiorum. Plants resistant to Sclerotinia sclerotiorumalso demonstrate some resistance to Botrytis cinerea.

Example 24 Soybeans are Made Resistant to Sudden Death Syndrome(Fusarium solani pisi Cutinase)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to F. solani pisi cutinase mRNA (GenBank locusK02640.1, SEQ ID NO: 48) Soybean plants are regenerated from transformedcells and successfully selected for conferred resistance to sudden deathsyndrome following inoculation with Fusarium solani glycines. Plantsresistant to Fusarium solani glycines show some increased resistance toA. nidulans.

Example 25 Soybeans are Made Resistant to Asian Rust (Phakopsorapachyrhizi Cutinase Homolog)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Phakopsora pachyrhizi conserved protein motif ofPhytophthora nicotianae and found in the Phakopsora pachyrhizi genome(GenBank locus AC149367.2, SEQ ID NO: 49). Based on homology withPhytophthora nicotianae cutinase, this conserved protein motif is likelya cutinase homologue and involved in penetrance of the soybean cutin.Soybean plants are regenerated from transformed cells and successfullyselected for conferred resistance to Asian rust following inoculationwith Phakopsora pachyrhizi.

Example 26 Soybeans are Made Resistant to Asian Rust (Phakopsorapachyrhizi rDNA)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to a ribosomal DNA sequence found in the Phakopsorapachyrhizi genome (GenBank locus AF333491, SEQ ID NO: 50). Soybeanplants are generated from transformed cells and successfully selectedfor conferred resistance to Asian rust following inoculation withPhakopsora pachyrhizi.

Example 27 Testing of Tobacco, Soybean, Potato and Corn forTransgenesis, Resistance to Phytophthora spp. and Resistance to AphidsTobacco

Mature tobacco plants produced in Example 5 by transformation withpVZA100 were tested for resistance against P. nicotianae. Three methodswere tested for inoculation with P. nicotianae. All gave satisfactoryresults, but as explained above, the toothpick method was more rigorousand more dependable. Generally about 20% of the TO plants transformedwith pVZA100 (initial transgenic generation) survived infection with P.nicotianae, whereas 0% of the wild type plants and those plantstransformed with pCAMBIA 1201 survived.

Surviving plants transformed with pVZA100 were grown to maturity insoil, and seed was collected for subsequent analysis. The seed progeny(=T1 generation) were grown on MS medium containing 30 ug/ml hygromycin,and the surviving plants again tested for resistance to P. nicotianae.

Surviving transformed plants also were grown to maturity in soil andseed collected for subsequent analysis. A group of seed progeny (=T2generation) of four different lines (lines #4, #23, #26 and #27;=different transformation events) of these plants were inoculated twicesuccessively to ensure that any survivors were resistant to P.nicotianae. In the first inoculation 192 of 442 plants were resistant(=43%). Upon reinoculation: 150 of 192 plants were resistant (=78%). Insummary, 150 of 442 plants were resistant (=34%). All of these 150resistant plants were also assayed for GUS activity and found to benegative.

Reactions by tobacco plants are shown in FIG. 11. Plant A showsreactions typical of wild type tobacco plants or those transformed withpCAMBIA1201. The plants are susceptible, there are major stem lesions,and the root system is essentially destroyed. Such plants usually die5-8 days after inoculation. Plant B is from Transgenic line 26 (seebelow). It is typical of those plants transformed with pVZA100. It washighly resistant to P. nicotianae and showed no symptoms other thansmall lesions on the tips of the upper roots. Such plants grow tomaturity and set fertile seed.

pVZA100 in Soybean:

Soybean plants were bombarded with pVZA100. PCR with the appropriateprimers demonstrated that 15 of 32 bombarded plants were transgenic.Transgenic plants were cultivated to produce seeds and seed plants weretested for resistance to P. sojae. As shown in FIG. 13, 43 of 495 seedplants tested were resistant to P. sojae (FIG. 13) whereas 100% of asimilar number of wild-type and pCAMBIA1201 transformed (i.e.“control-transformed”) plants died.

pVZA100 in Potato

Potato plants were bombarded with pVZA100. PCR with the appropriateprimers demonstrated that 15 of 18 bombarded plants were transgenic.Transgenic plants are tested for resistance to P. infestans. 100% ofthese plants are resistant as compared to 0% resistance in wild-type orcontrol-transformed plants.

pVZA100 in Corn

Corn plants were bombarded with pVZA100. PCR with the appropriateprimers demonstrated that 33 of 44 bombarded plants were transgenic.Transgenic plants are cultivated to produce seeds and seed plant aretested for resistance to Pythium graminicola. 25% of the F1 plantstested are resistant as compared to 0% resistance in wild-type orcontrol-transformed plants.

The above results demonstrate that plants useful in the presentinvention include monocots and dicots.

Similarly, corn plants are transformed with pVZA100 and tested andillustrate cross resistance to Phytophthora and Peronospora.

pVZA200 in Potato

Potato plants were bombarded with pVZA200 (Myzus persicae Cathepsin B).PCR with the appropriate primers demonstrated that 7 of 12 bombardedplants were transgenic. Transgenic plants are tested for resistance toaphids (Myzus persicae). 80% of such plants, compared to 0% of controlor wildtype plants, are resistant.

Aphids after feeding on transformed plants are examined. It isdetermined that silencing cathespin B in aphids according to the presentinvention not only protects the plant (i.e. causes aphid resistance inplants) but also reduces the aphid population.

According to the present invention and this example, insect resistanceis conferred to plants through transformation with a silencing constructcontaining an insect gene such as Cathepsin B.

pVZA300 in Potato

Potato plants were bombarded with pVZA300, a construct containing senseand antisense sequences homologous and complementary to Cathepsin B fromMyzus persicae and elicitin INF1 of Phytophthora infestans. PCR with theappropriate primers demonstrated that 15 of 18 bombarded plants weretransgenic. Such transgenic plants are tested for resistance to aphids(Myzus persicae) and to Phytophthora infestans.

In one set of experiments, one group of transformed potato plants arechallenged with aphids only and one group is challenged withPhytophthora infestans only.

For the aphid challenged plants, 80% of such plants tested are resistantas compared to 0% resistance in wild-type or control-transformed plants.

For the Phytophthora infestans challenged plants, 75% of such plantstested were resistant as compared to 0% resistance in non-transformed orcontrol plants.

In another set of experiments, similarly transformed and non-transformedpotato plants are challenged with aphids and Phytophthora infestans atthe same time. 60% of the transgenic plants tested are resistant to bothpests as compared to 0% resistance in wild-type or control-transformedplants.

After feeding on transformed plants, Phytophthora infestans is isolatedand cultured. Such cultures either grow very slowly or die. Microscopicexamination reveals that the hyphae are severely malformed.

Moreover, silencing rRNA according to the present invention furtherillustrates an embodiment whereby the pathogen population in the soil isdramatically reduced.

pVZA300 in Soybeans

Soybean plants were bombarded with pVZA300 (a construct containing senseand antisense sequence homologous and complementary to Cathepsin B fromMyzus persicae and elicitin INF1 of Phytophthora infestans). PCR withthe appropriate primers demonstrated that 60% of bombarded plants aretransgenic. Transgenic plants are cultivated to produce seeds and seedplants are grown. Such F1 plants are tested for resistance to aphids(Myzus persicae) and to Phytophthora sojae.

In one set of experiments, one group of transformed soybean plants arechallenged with aphids only and one group is challenged with Myzuspersicae only.

For the aphid challenged plants, 70% of the F1 plants tested areresistant as compared to 0% resistance in wild-type orcontrol-transformed plants.

For the Phytophthora sojae challenged plants, 15% of the F1 plantstested are resistant as compared to 0% resistance in wildtype and incontrol transformed plants.

In another set of experiments, transformed and non-transformed soybeansplants are challenged with aphids and Phytophthora sojae at the sametime. 10% of the F1 plants tested are resistant to both pests ascompared to 0% resistance in wild-type or control-transformed plants.

According to the present invention and these examples, insect resistanceand fungus resistance is conferred in a plant (both for moncot and dicotspecies) by a single heterologous polynucleotide comprising a pluralityof genes (i.e. by gene stacking). Moreover, a surprising level of pestresistance is conferred in plants by silencing genes from more than onepest.

After feeding on transformed plants, Phytophthora sojae is isolated andcultured. Such cultures either grow very slowly or die. Microscopicexamination reveals that the hyphae are severely malformed.

pVZA400 in Soybeans

Soybean plants were bombarded with pVZA400 comprising antisense andsense sequences homologous and complementary to the rRNA gene ofPhytophthora infestans. PCR with the appropriate primers demonstratedthat 12 of 18 bombarded plants were transgenic. Transgenic plants arecultivated to produce seeds and seed plants are tested for resistance toPhytophthora sojae. 15% of the F1 plants that are tested are resistantas compared to 0% resistance in non-transformed or control transgenicplants.

After feeding on transformed plants, Phytophthora sojae is isolated andcultured. Such cultures either grow very slowly or die. Microscopicexamination reveals that the hyphae are severely malformed.

Next, wild-type soybeans are challenged with Phytophthora sojae culturesderived from the pVZA400-transformed plants. Such P. sojae cultures arenon pathogenic in comparison to sojae cultures which are derived fromwild-type or control-transformed soybeans.

The above results illustrate the usefulness of silencing of rRNA in apest (e.g. fungi) and further illustrates cross resistance to a pestspecies other than the pest from which the pest pathogenicity gene wasconstructed.

After feeding on transformed plants, Phytophthora sojae is isolated andcultured. Such cultures either grow very slowly or die. Microscopicexamination reveals that the hyphae are severely malformed.

Moreover, silencing rRNA according to the present invention furtherillustrates an embodiment whereby the pathogen population in the soil isdramatically reduced.

pVZA400 in Potato

Potato plants were bombarded with pVZA400 comprising antisense and sensesequences mologous and complementary to the rRNA gene of Phytophthorainfestans. PCR with the appropriate primers demonstrated that 6 of 12bombarded plants were transgenic. Transgenic plants were cultivated andtested for resistance to Phytophthora infestans. These plants weretested and showed resistance in 100% of these plants as compared to 0%resistance in wild-type or control-transformed plants.

After feeding on transformed plants, Phytophthora infestans is isolatedand cultured. Such cultures either grow very slowly or die. Microscopicexamination reveals that the hyphae are severely malformed.

The above results illustrate the usefulness of a single silencingconstruct of rRNA in conferring pest resistance in a plurality of plantsto a pest (e.g. fungi).

Example 28 Potato is Made Resistant to Tuber Rot (Fusarium sambucinumTranslation Elongation Factor 1 Alpha)

Potato host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to a Fusarium sambucinum translation elongationfactor 1 alpha DNA sequence (GenBank locus AJ543605, SEQ ID NO: 51).Potato plants are generated from transformed cells and successfullyselected for conferred resistance to Fusarium sambucinum tuber rot.

Example 29 Tomato is Made Resistant to Late Blight (Phytophthorainfestans Elicitin Gene)

Tomato host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to an elicitin gene sequence found in thePhytophthora infestans genome (GenBank locus AY766228, SEQ ID NO: 52).Tomato plants are generated from transformed cells and successfullyselected for conferred resistance to late blight of tomato followinginoculation with Phytophthora infestans.

Example 30 Canola is Made Resistant to Stem Rot (Sclerotiniasclerotiorum RNA Polymerase Subunit)

Canola host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to a Sclerotinia sclerotiorum partial rpb2 gene forRNA polymerase II second largest subunit, exon 1, strain 484 1096 bp(GenBank locus AJ745716, SEQ ID NO: 53). Canola plants are generatedfrom transformed cells and successfully selected for conferredresistance to stem rot following inoculation with Sclerotiniasclerotiorum.

Example 31 Cotton is Made Resistant to Cotton Wilt (Fusarium solani pisiCutinase Homolog)

Cotton host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to a cutinase gene from F. solani pisi (GenBanklocus K02640.1, SEQ ID NO: 48). This sequence is picked herein becauseof the homology of genes within the genus Fusarium. Cotton plants aregenerated from transformed cells and successfully selected for conferredresistance to cotton wilt following inoculation with Fusarium oxysporum.

Example 32 Corn is Made Resistant to Mycotoxins (F. solani pisiCutinase)

Corn host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to a cutinase gene from an F. solani pisi (GenBanklocus K02640.1, SEQ ID NO: 48). This sequence is picked herein becauseof the homology of genes within the genus Fusarium. Corn plants aregenerated from transformed cells and selected for increased resistanceto mycotoxins following inoculation with Fusarium moniliforme.

Example 33 Corn is Made Resistant to Stalk Rot (Stenocarpella maydisrDNA)

Corn host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Stenocarpella maydis rRNA (GenBank locusAY332489, SEQ ID NO: 54). This sequence is picked herein because of thehomology of genes within the genera Stenocarpella and Fusarium. Cornplants are generated from transformed cells and selected for increasedresistance to stalk rot following inoculation with Stenocarpella maydisand several Fusarium spp.

Example 34 Corn is Made Resistant to Leaf Blight (Cercospora zeae-maydis18s rDNA)

Corn host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to 18S ribosomal RNA from a Cercospora zeae-maydisGroup II (GenBank locus AF291710, SEQ ID NO: 55). Corn plants aregenerated from transformed cells and successfully selected for conferredresistance to leaf blight following inoculation with Cercosporazeae-maydis.

Example 35 Potato is Made Resistant to Green Peach Aphid (Myzus persicaeCathepsin B Protease)

Potato host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Cathepsin B protease from Myzus persicae(GenBank locus AY702822, SEQ ID NO: 56). Potato plants are generatedfrom transformed cells and successfully selected for conferredresistance to green peach aphids following inoculation with Myzuspersicae.

Example 36 Potato is Made Resistant to Colorado Potato Beetle(Leptinotarsa decemlineata Cysteine Protease)

Potato host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to cysteine protease from Leptinotarsa decemlineata(GenBank locus AY159377, SEQ ID NO: 57). Potato plants are generatedfrom transformed cells and successfully selected for conferredresistance to Colorado potato beetle following inoculation withLeptinotarsa decemlineata. Such regenerated potatoes demonstrateincreased resistance to each of the following: Callosobruchus maculates,Diabrotica virgifera virgifera, Acanthoscelides obtectus, Helicoverpaarmigera, Leptinotarsa decemlineata, Anthonomus grandis, Triatomainfestans, Tenebrio molitor, Toxoptera citricida, Aphis gossypii,Pandalus borealis, Myzus persicae, Delia radicum, Sarcophaga peregrine,Sitophilus zeamais, Boophilus microplus, Hypera postica, Frankliniellaoccidentalis, and H. americanus.

Example 37 Potato is Made Resistant to Verticillium Wilt (V. dahliaerDNA)

Potato host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Verticillium dahliae rDNA (GenBank locusAF108478, SEQ ID NO: 58). Potato plants are generated from transformedcells and successfully selected for conferred resistance to Verticilliumwilt following inoculation with Verticillium dahliae.

Example 38 Soybeans are Made Resistant to Downy Mildew (Peronosporaberteroae rDNA)

Soybean host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to internal transcribed spacer 1 partial sequenceof 5.8S ribosomal RNA found in the Peronospora berteroae genome (GenBanklocus AY531450, SEQ ID NO: 59). Soybean plants are generated fromtransformed cells and successfully selected for conferred resistance todowny mildew following inoculation with Peronospora manchurica. Suchselected soybeans also have increased resistance to Hyaloperonosporaspecies (e.g. H. camelinae, H. niessleana, H. parasitica, and H.thlaspeos-perfoliati) and Peronospora species (e.g. P. arabidopsidis, P.arabis-alpinae, P. buniadis, P. cardaminopsidis, P. cochleariae, P.dentariae, P. galligena, P. hesperidis, P. iberidis, P. isatidis, P.nesleae, P. sisymbrii-loeselii, P. sisymbrii-offcinalis, P.sisymbrii-sophiae, P. thlaspeos-alpestris, and P. thlaspeos-arvensis).

Example 39 Potato is Made Resistant to Potato Root Knot Nematode(Meloidogyne chitwoodi Cytochrome Oxidase II)

Potato host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to a sequence found in the Meloidogyne chitwoodigenome (i.e. cytochrome oxidase subunit II gene, (GenBank locusAY757882, SEQ ID NO:60). Potato plants are generated from transformedcells and successfully selected for conferred resistance to potato rootknot following inoculation with Meloidogyne chitwoodi. Such selectedpotato plants are also found to be resistant to Bombyx mandarina, Bombyxmori, Chilo tumidicostalis, Hemileuca, Hydrotaea aenescens, Meloidogynehapla, Meloidogyne partityla, Oecetis avara, Papilio aegeus, Papiliodemoleus, Papilio erithonioides, Papilio hipponous, Papilio oribazus,Papilio protenor, Papilio troilus, Papilio xuthus, Sabethes cyaneus, andSamia cynthia ricini.

Example 40 Soybeans are Made Resistant to Nematode Infestation(Pratylenchus scribneri and Heterodera glycines Ribosomal RNA Genes)

The present invention can be used to produce host plants with increasedresistance to additional nematodes. Nematode polynucleotide sequencesthat are known and are searchable in public databases such as theNCBI/NIH GenBank demonstrate conserved nucleotide motifs among differentnematode genera. Conserved nucleotide motifs strongly suggest that thesesequences are associated with viability and/or parasitism and arefunctionally conserved and expressed in both Meloidogyne incognita(root-knot nematode) and Globodera rostochiensis and Globdera pallids(potato cyst nematodes), and also in the well studied Heteroderaglycines and Caenorhabditis elegans. Thus, the use of these sequencesand variants thereof, is advantageous because such RNAi can be designedto have broad RNAi specificity and are thus useful for controlling alarge number of plant parasitic nematodes in planta. Because genesidentified herein are associated with nematode survival and/orparasitism, RNAi inhibition of these genes (arising from contactingnematodes with compositions comprising RNAi molecules such as, forexample, by allowing nematodes to feed on plants transformed as taughtherein) prevents and/or reduces parasitic nematode growth, development,and/or parasitism.

Nematode feeding is a useful indicator of the effectiveness of themethods of the present invention. The Pratylenchus scribneri in vitrofeeding assay uses a corn root exudate as a feeding stimulus and boththe red dye Amaranth, or potassium arsenate, as feeding indicators.Feeding is confirmed after seven days by the presence of red stainedintestinal cells in live worms exposed to the Amaranth or death of wormsexposed to arsenate. P. scribneri can be cultured on wild type roots ofcorn, rice and Arabidopsis, and on Agrobacterium rhizogenes-inducedhairy roots of sugar beet and tomato. P. scribneri is very valuable inevaluating transgenic hairy roots (ideally developed by use ofAgrobacterium rhizogenes strains known in the art as transformationvectors) because of the non-specific feeding of these worms.

Corn, rice, tomato, and sugar beets are transformed with a heterologouspolynucleotide containing complementary sense and antisense sequences ofP. scribneri ribosomal RNA (GenBank locus SEQ ID NO:61). P. scribneri istested on a feeding assay using transformed and wild type corn, rice,tomato, and sugar beets. Transformed plants demonstrate increasedresistance to P. scribneri.

Soybeans are transformed with a heterologous polynucleotide containingcomplementary sense and antisense sequences of Heterodera glycinesribosomal RNA (GenBank locus AY667456, SEQ ID NO: 62). Heteroderaglycines is tested on a feeding assay using transformed and wild typesoybeans. Transformed soybeans demonstrate increased resistance toHeterodera glycines.

Example 41 Various Plants are Made Resistant to Infection by VariousPhytophthora spp. (Phytophthora nicotianae Cutinase)

In one embodiment, the pVZA100 heterologous polynucleotide (Example 3)is used to transform a variety of plants to confer resistance to variousPhytophthora species. For example, resistance is conferred againstPhytophthora spp.-induced leaf and stem infections when any ofChrysalidocarpus palm, Catharanthus, Dendrobium, poinsettia, impatiens,sage, spathiphyllum, or onion is transformed with the pVZA100 (SEQ IDNO: 5). Resistance is conferred against Phytophthora spp.-induced rootrot when any of poinsettia, tomato, pineapple, or anthurium istransformed with the pVZA100.

Resistance is conferred against Phytophthora spp.-induced crown andcollar rots when any of watermelon, protea, or African violet istransformed with the pVZA100.

Resistance is conferred against Phytophthora spp.-induced fruit rotswhen any of tomato, papaya, or eggplant is transformed with the pVZA100.

Example 42 Agave is Made Resistant to Agave Root Rot (Fusarium solanipisi Cutinase)

Agave host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to a cutinase gene from Fusarium solani pisi(GenBank locus K02640.1, SEQ ID NO: 48) This sequence is picked hereinbecause of the homology of genes within the genus Fusarium. Agave plantsare generated from transformed cells and successfully selected forconferred resistance to Agave rot following inoculation with Fusariumoxysporum

Example 43 Rice is Made Resistant to Blast Disease (Magnaporthe griseaRNA Polymerase II)

Rice host cells are transformed with a heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to a Magnaporthe grisea RNA polymerase II sequenceof (GenBank locus XM 362268, SEQ ID NO: 63). Transformation wasaccomplished by electroporation or Agrobacterium tumefaciens. Riceplants are generated from transformed cells and successfully selectedfor conferred resistance to rice blast disease following inoculationwith Magnaporthe grisea.

Example 44 Various Plants are Made Resistant to Phytophthora-InducedRoot Disease (Phytophthora citricola Cutinase)

Various plants cells including those from Rhododendron (e.g. Rh.catawbiense, Rh. simsii, Rh. Catawbiense), Erica sp., Calluna sp.,avocado trees, citrus trees, and hops are transformed with theheterologous polynucleotide pVZA100 (Example 3). This construct waschosen because of the homology between Phytophthora nicotianae SEQ IDNO: 14 and Phytophthora citricola SEQ ID NO: 34. Host plants aregenerated from transformed cells and successfully selected for conferredresistance to disease following inoculation with Phytophthora citricola.

Example 45 Barley is Made Resistant to Barley Leaf Rust (Puccinia hordei18S Ribosomal RNA)

Barley cells are transformed with the heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Puccinia hordei 18S rRNA (GenBank locusAY125412, SEQ ID NO:64). Host plants are generated from transformedcells and successfully selected for conferred resistance to barley leafrust following inoculation with Puccinia hordei.

Example 46 Barley is Made Resistant to Downy Mildew (Pseudoperonosporahumuli rDNA)

Barley cells are transformed with the heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to sequences from Pseudoperonospora humuli isolateHV 148 internal transcribed spacer 1, partial sequence; 5.8S ribosomalRNA (GenBank locus AY198305, SEQ ID NO: 65). Host plants are generatedfrom transformed cells and successfully selected for conferredresistance to downy mildew following inoculation with Pseudoperonosporahumuli.

Example 47 Barley is Made Resistant to Gray Mold Disease (BotrytisCinerea Cytochrome P450 Monoxygenase)

Barley cells are transformed with the heterologous polynucleotidesimilar to pVZA100

(Example 3) except that the sense and antisense sequences correspond tosequences from Botrytis cinerea for aba2 gene for cytochrome P450monoxygenase, exons 1-5 (GenBank locus AJ851088, SEQ ID NO: 66). Hostplants are generated from transformed cells and successfully selectedfor conferred resistance to gray mold disease following inoculation withBotrytis cinerea.

Example 48 Tomato is Made Resistant to Tomato Speck Disease (Pseudomonassyringae rDNA)

Tomato cells are transformed with the heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Pseudomonas syringae ribosomal RNA (GenBanklocus AY342210, SEQ ID NO: 67). Host plants are generated fromtransformed cells and are successfully selected for conferred resistanceto tomato speck disease following inoculation with Pseudomonas syringae.

Example 49 Tomato is Made Resistant to Bacterial Wilt (Clavibactermichiganense michiganense Cel A Gene)

Tomato cells are transformed with the heterologous polynucleotidesimilar to pVZA100 (Example 3) except that the sense and antisensesequences correspond to Clavibacter michiganense michiganense Cel A gene(GenBank locus AY007311, SEQ ID NO: 68). Host plants are generated fromtransformed cells and are successfully selected for conferred resistanceto bacterial wilt following inoculation with Clavibacter michiganensemichiganense.

Example 50 Corn is Made Resistant to Goss's Bacterial Wilt and Blight(Clavibacter michiganense Endo β-Glucosidase Gene)

Corn cells are transformed with the heterologous polynucleotide similarto pVZA100 (Example 3) except that the sense and antisense sequencescorrespond to Clavibacter michiganense gene for endo β-glucosidase(GenBank locus X62582, SEQ ID NO: 69). Host plants are generated fromtransformed cells and are successfully selected for conferred resistanceto Goss's bacterial wilt and blight (leaf freckles and wilt) followinginoculation with Clavibacter michiganense.

Example 51 A Silencing Construct Method to Protect Soybeans Against Oneor Two Fungal Diseases

A soybean host plant cell is transformed with either of the genesilencing constructs of FIG. 10 and mature plants are generatedtherefrom. Mature plants are screened for resistance to two major fungaldiseases, namely soybean root and stem rot caused by Phytophthora sojaeand Asian rust of soybean caused by Phakopsora pachyrhizi. Plants areidentified that are resistant to each and to both of these two diseases.Those plants transformed with the upper construct are resistant tosoybean root and stem rot. Those plants transformed with the middleconstruct are resistant to Asian rust of soybean. Those plantstransformed with the lower construct are resistant to both soybean rootand stem rot and Asian rust of soybean.

Example 52 A Polycistronic Silencing Construct Method to ProtectSoybeans Against Three Fungal Diseases

A soybean host plant cell is transformed with a polycistronic genesilencing construct of FIG. 9 and mature plants are generated therefrom.Mature plants are screened for resistance to three major fungaldiseases, namely soybean root and stem rot caused by Phytophthora sojae,soybean sudden death syndrome caused by Fusarium solani f. sp. glycines,and Asian rust of soybean caused by Phakopsora pachyrhizi. Plants areidentified that are resistant to each and all of these three diseases.

Example 53 Depathogenesis of Phytophthora Species by Feeding onTransgenic Plants or by Direct Transformation with pVZA100, pVZA300 orpVZA400

Tobacco host cells were transformed with the silencing construct pVZA100(SEQ ID NO: 5) and plants were generated therefrom. Such transformedplants were highly resistant to P. nicotianae. Typical reactions bytobacco plants are shown in FIG. 11. Plant A shows reactions typical ofwild type tobacco plants or those transformed with pCAMBIA1201. Theplants are susceptible, there are major stem lesions, and the rootsystem is essentially destroyed. Such plants usually die 5-8 days afterinoculation. Plant B is from Transgenic line 26 (see below). It istypical of those plants transformed with pVZA100. It is highly resistantto P. nicotianae and shows no symptoms other than small lesions on thetips of the upper roots. Such plants grow to maturity and set fertileseed.

When P. nicotianae was re-isolated from the restricted lesions on theresistant plant and re-tested on wild type plants, it was no longerpathogenic. Isolation of RNA from this fungus and hybridization with theradioactive cutinase probe as above in Example 14 indicates the presenceof siRNAs for the cutinase gene in P. nicotianae, thereby explaining theloss of pathogenicity. In FIG. 12, lane 1 again shows the 35 ntoligonucleotides VZA 3F (SEQ ID NO: 3) and VZA 4R (SEQ ID NO: 4) servingas the molecular size control, lane 2 shows the 620 nt messenger RNAfrom the wild type fungus, lane 3 shows the siRNAs from a silencedtransgenic tobacco plant, and lane 4 shows the siRNAs from thenonpathogenic fungus isolated from the resistant transgenic tobacco.

As shown in Example 13, transforming species of a pest directly withsilencing constructs or by feeding pest species on plants transformedwith silencing constructs had major effects on the pathogenicity, growthand survival of the pest (in this example, the fungi). These resultssupport directly or indirectly the utility of this embodiment of thepresent invention to minimize crop losses to pests and to enhanceagricultural productivity.

Example 54 Depathogenesis: Watermelon Transformed with a CutinaseSilencing Heterologous Polynucleotide (F. Oxysporum Cutinase)

In Florida and Georgia watermelons can be planted in a field for onlyone year. The farmer must then wait 6 or 7 years to use that field againbecause a single year of use results in the high build up of pathogenicisolates of Fusarium oxysporum. To use a field in successive years, thefarmer must resort to expensive and severe annual treatments with methylbromide or other potent chemicals.

Watermelons are regenerated from a host plant cell transformed with aheterologous polynucleotide similar to pVZA100 (Example 3) except thatthe sense and antisense sequences correspond to the cutinase gene in F.oxysporum. F. oxysporum infects the watermelon and the F. oxysporumcutinase gene is silenced. Other watermelon species (not transformedaccording to the present invention) are planted within six feet of theregenerated (transgenic) watermelon. Such other watermelon species areinfected by F. oxysporum to a lesser extent than would be predicted wereit not for the cultivation of the regenerated (transgenic) watermelonsadjacent to them.

Example 55 Broad Based Plant Resistance and Depathogenesis withRibosomal RNA Silencing Heterologous Polynucleotides

Optionally, to effectively implement depathogenesis, a broad-based orcross generic protection against several species of pathogenic fungi canbe obtained. This can be accomplished by utilizing silencing constructscontaining ribosomal DNA (rDNA) genes. The rDNA sequences of fungalpathogens are similar enough to function in the control of closelyrelated fungal species, but are sufficiently dissimilar to permitdifferentiation among groups of fungi, thereby enabling the selectivetargeting of specific groups of fungal pathogens with single or multipleor polycistronic gene silencing constructs.

This approach is illustrated in Table 13. The 18S rDNA sequences ofPhytophthora sojae and Phakopsora pachyrhizi were “blasted” against thedata in the NCBI/NIH GenBank. The Phytophthora sojae sequence showedstrong homology (100 to 93%) to the sequences of 16 additional closelyrelated fungal species, but it did not show significant homology to thatof Phakopsora pachyrhizi. Conversely, the 18S rDNA sequence ofPhakopsora pachyrhizi showed strong homology (100 to 93%) to thesequences of 19 additional closely related fungal species, but it didnot show significant homology to that of Phytophthora sojae. Therefore,individual gene silencing constructs can be developed in accord with theteachings herein to control the group of fungi related to Phytophthorasojae or to Phakopsora pachyrhizi, or alternatively a polycistronic genesilencing construct can be developed to control both groupssimultaneously.

TABLE 13 GenBank sequences ribosomal DNA producing significantalignments with target species Phytophthora sojae 18s ribosomal DNAPhakopsora pachyrhizi 18s ribosomal DNA The identities of the sequencesranges from The identities of the sequences ranges from 100 to 93% over129 nucleotides. 100 to 93% over 89 nucleotides. Fungus Species = (17)(bits) Fungus Species = (18) (bits) Phytophthora sojae 278 Phakopsorapachyrhizi 278 Phytophthora vignae 194 Phakopsora meibomiae 153Phytophthora drechsleri 174 Uromyces aemulus 131 Phytophthora cinnamomi174 Puccinia striiformis 123 Plasmopara viticola 168 Pandora neoaphidis121 Phytophthora melonis 167 Puccinia allii 119 Phytophthora cryptogea165 Piromyces sp. 119 Phytophthora pistaciae 161 Dioszegia sp. 117Hyaloperonospora parasitica 161 Bullera sp. T 117 Phytophthoraniederhauserii 161 Dioszegia crocea 117 Phytophthora sinensis 153Cadophora sp. 117 Phytophthora cajani 153 Dioszegia hungarica 117Pythium insidiosum 153 Phialophora sp. 117 Phytophthora palmivora 151Anaeromyces sp. 117 Phytophthora ramorum 149 Phialophora melinii 117Peronospora corydalis 149 Puccinia triticina 115 Pythium rostratum 141Neocudoniella radicella 115 Pinctada nigra 115 Peziza vesiculosa 115Phillipsia domingensis 115

Example 56 Control of Multiple Plant Pests Using Multiple SilencingHeterologous Polynucleotides

Plants are often attacked by a multiplicity of pests. Optionally, it maybe useful to control several pests on the same plant. Methods for genestacking to control multiple pests and the useful gene sequences (forexample, genes encoding ribosomal RNAs, cutinases, cathepsins and otheressential enzymes and proteins) are taught herein. A nonlimiting exampleis soybeans and the simultaneous control of Phytophthora root and stemrot, Asian rust, the soybean cyst nematode, sudden death syndrome andaphids. Exemplary genes to stacked for this purpose include SEQ ID NO:14 for Phytophthora root and stem rot, SEQ ID NO: 50 for Asian rust, SEQID NO: 62 for the soybean cyst nematode, SEQ ID NO: 48 for sudden deathsyndrome, and SEQ ID NO: 56 for aphids.

Similarly, corn can be made resistant to root and stalk rots,mycotoxins, leaf blights, stalk and ear borers, aphids and nematodes.Similarly, cotton can be made resistant to boll rot, Verticillium andFusarium wilts, seedling blights, damping off, boll weevil, bollworms,aphids, and loopers. Similarly, canola can be made resistant to blackleg, stem rot, black spot, damping off and seedling blights, fleabeetles, weevils, and aphids.

Similarly, wheat can be made resistant to leaf and stem rust, headblight, Septoria blotches, take-all, smuts, powdery and downy mildew,aphids and nematodes. Similarly, tomato can be made resistant to lateblight, anthracnose, gray mold, Verticillium wilt, nematodes, andaphids. Similarly, potato can be made resistant to late blight, earlyblight, Verticillium wilt, nematodes, Colorado potato beetle, andaphids.

All references cited herein are incorporated by reference herein for allthat they teach and for all purposes, to the extent they are notinconsistent with the explicit teachings herein. It is to be understoodthat, while the invention has been described in conjunction with thedetailed description thereof, the foregoing description is intended toillustrate and not limit the scope of the invention, which is defined bythe scope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims.

The Sequence Listings are incorporated herein by reference.

1-25. (canceled)
 26. A method of conferring fungal resistance to a plantcomprising expressing in the plant: (a) an antisense sequence having atleast about 80% homology to a stretch of at least about 20 nucleotidesof at least one sequence selected from the group consisting of SEQ IDNOs: 8, 43, 44, 46, 50, 54, 55, 58, 59 and 65; (b) a sense sequencesubstantially complementary to the antisense sequence, wherein atranscript of the antisense sequence and a transcript of the sensesequence are capable of hybridizing to form a double-stranded region;and whereby the plant is resistant to a fungus expressing at least oneribosomal RNA gene having at least about 80% homology to the antisensesequence.
 27. The method of claim 26, wherein the antisense sequence ishomologous to a stretch of at least about 20 nucleotides of at least oneadditional sequence selected from the group consisting of SEQ ID NOs: 8,43, 44, 46, 50, 54, 55, 58, 59 and 65, whereby the plant is resistant toa fungus expressing at least one ribosomal RNA gene having at leastabout 80% homology to the at least one additional sequence.
 28. Themethod of claim 27, wherein the antisense sequence has at least about80% homology to the stretch of at least about 20 nucleotides of the atleast one additional sequence.
 29. The method of claim 26, wherein thetranscript of the antisense sequence and the transcript of the sensesequence form a double stranded region.
 30. The method of claim 27,wherein the transcript of the antisense sequence and the transcript ofthe sense sequence form a double stranded region.
 31. The method ofclaim 28, wherein the transcript of the antisense sequence and thetranscript of the sense sequence form a double stranded region.
 32. Themethod of claim 26, further comprising expressing in the plant: (c) asecond antisense sequence having at least about 80% homology to astretch of at least about 20 nucleotides of a second sequence selectedfrom the group consisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58,59 and 65; and (d) a second sense sequence substantially complementaryto the second antisense sequence; wherein a transcript of the secondantisense sequence and a transcript of the second sense sequence arecapable of hybridizing to form a double-stranded region; and whereby theplant is resistant to a fungus expressing at least one ribosomal RNAgene having at least about 80% homology to the second antisensesequence.
 33. The method of claim 32, wherein the transcript of thesecond antisense sequence and the transcript of the second sensesequence form a double stranded region.
 34. A transgenic plant that isresistant to fungus, the transgenic plant comprising: (a) an antisensesequence having at least about 80% homology to a stretch of at leastabout 20 nucleotides of at least one sequence selected from the groupconsisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58, 59 and 65; (b)a sense sequence substantially complementary to the antisense sequence,wherein a transcript of the antisense sequence and a transcript of thesense sequence are capable of hybridizing to form a double-strandedregion; and whereby the plant is resistant to a fungus expressing atleast one ribosomal RNA gene having at least about 80% homology to theantisense sequence.
 35. The transgenic plant of claim 34, wherein theantisense sequence is homologous to a stretch of at least about 20nucleotides of at least one additional sequence selected from the groupconsisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58, 59 and 65,whereby the plant is resistant to a fungus expressing at least oneribosomal RNA gene having at least about 80% homology to the at leastone additional sequence.
 36. The transgenic plant of claim 35, whereinthe antisense sequence has at least about 80% homology to the stretch ofat least about 20 nucleotides of the at least one additional sequence.37. The transgenic plant of claim 34, wherein the transcript theantisense sequence and the transcript of the sense sequence form adouble stranded region.
 38. The transgenic plant of claim 35, whereinthe transcript the antisense sequence and the transcript of the sensesequence form a double stranded region.
 39. The transgenic plant ofclaim 36, wherein the transcript the antisense sequence and thetranscript of the sense sequence form a double stranded region.
 40. Thetransgenic plant of claim 34, further comprising: (c) a second antisensesequence having at least about 80% homology to a stretch of at leastabout 20 nucleotides of a second sequence selected from the groupconsisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58, 59 and 65; and(d) a second sense sequence substantially complementary to the secondantisense sequence; wherein a transcript of the second antisensesequence and a transcript of the second sense sequence are capable ofhybridizing to form a double-stranded region; and whereby the plant isresistant to a fungus expressing at least one ribosomal RNA gene havingat least about 80% homology to the second antisense sequence.
 41. Thetransgenic plant of claim 40, wherein the transcript the secondantisense sequence and the transcript of the second sense sequence forma double stranded region.